BACKGROUND OF THE INVENTION
[0001] The present invention relates generally to biosynthetic processes, and more specifically
to organisms having toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate,
(2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol
or 1,3-butadiene biosynthetic capability.
[0002] Toluene is a common solvent that has replaced benzene due to benzene's greater carcinogenicity
and is an industrial feedstock and used in the manufacture of TNT, polyurethane foam,
benzaldehyde and benzoic acid. Toluene is a byproduct in the manufacture of gasoline
and exists in small concentrations in crude oil.
[0003] Benzene is often used as an intermediate to make other chemicals. Its most widely-produced
derivatives include styrene, which is used to make polymers and plastics, phenol for
resins and adhesives, via cumene, and cyclohexane, which is used in the manufacture
of Nylon. Benzene is also used to make some types of rubbers, lubricants, dyes, detergents,
drugs, explosives, napalm and pesticides. Benzene production in the petroleum industry
is made by various energy intensive processes including, catalytic reforming, toluene
hydrodealkylation, toluene disproportionation, and steam cracking.
[0004] Styrene is the precursor to polystyrene and numerous copolymers. Styrene based products
include, acrylonitrite 1,3-butadiene styrene (ABS), styrene-1,3-butadiene (SBR) rubber,
styrene-1,3-butadiene latex, SIS (styrene-isoprene-styrene), S-EB-S (styrene-ethylene/butylene-styrene),
styrene-divinylbenzene (S-DVB), and unsaturated polyesters. These materials are used
in rubber, plastic, insulation, fiberglass, pipes, automobile and boat parts, food
containers, and carpet backing.
[0005] Styrene is most commonly produced by the catalytic dehydrogenation of ethylbenzene.
Ethylbenzene is mixed in the gas phase with 10-15 times its volume in high-temperature
steam, and passed over a solid catalyst bed. Most ethylbenzene dehydrogenation catalysts
are based on iron(III) oxide, promoted by several percent potassium oxide or potassium
carbonate. Steam serves several roles in this reaction. It is the source of heat for
powering the endothermic reaction, and it removes coke that tends to form on the iron
oxide catalyst through the water gas shift reaction. The potassium promoter enhances
this decoking reaction. The steam also dilutes the reactant and products, shifting
the position of chemical equilibrium towards products. A typical styrene plant consists
of two or three reactors in series, which operate under vacuum to enhance the conversion
and selectivity. Typical per-pass conversions are ca. 65% for two reactors and 70-75%
for three reactors.
[0006] Over 25 billion pounds of 1,3-butadiene (or just butadiene or BD) are produced annually
and is applied in the manufacture of polymers such as synthetic rubbers and ABS resins,
and chemicals such as hexamethylenediamine and 1,4-butanediol. 1,3-butadiene is typically
produced as a by-product of the steam cracking process for conversion of petroleum
feedstocks such as naphtha, liquefied petroleum gas, ethane or natural gas to ethylene
and other olefins. The ability to manufacture 1,3-butadiene from alternative and/or
renewable feedstocks would represent a major advance in the quest for more sustainable
chemical production processes
[0007] One possible way to produce 1,3-butadiene renewably involves fermentation of sugars
or other feedstocks to produce diols, such as 1,4-butanediol or 1,3-butanediol, which
are separated, purified, and then dehydrated to 1,3-butadiene in a second step involving
metal-based catalysis. Direct fermentative production of 1,3-butadiene from renewable
feedstocks would obviate the need for dehydration steps and 1,3-butadiene gas (bp
-4.4°C) would be continuously emitted from the fermenter and readily condensed and
collected. Developing a fermentative production process would eliminate the need for
fossil-based 1,3-butadiene and would allow substantial savings in cost, energy, and
harmful waste and emissions relative to petrochemically-derived 1,3-butadiene.
[0008] 2,4-Pentadienoate is a useful substituted butadiene derivative in its own right and
a valuable intermediate en route to other substituted 1,3-butadiene derivatives, including,
for example, 1-carbamoyl-1,3-butadienes which are accessible via Curtius rearrangement.
The resultant N-protected-1,3-butadiene derivatives can be used in Diels alder reactions
for the preparation of substituted anilines. 2,4-Pentadienoate can be used in the
preparation of various polymers and co-polymers.
[0009] Terephtalate (also known as terephthalic acid and PTA) is the immediate precursor
of polyethylene terephthalate (PET), used to make clothing, resins, plastic bottles
and even as a poultry feed additive. Nearly all PTA is produced from
para-xylene by oxidation in air in a process known as the Mid Century Process. This oxidation
is conducted at high temperature in an acetic acid solvent with a catalyst composed
of cobalt and/or manganese salts. Para-xylene is derived from petrochemical sources
and is formed by high severity catalytic reforming of naphtha. Xylene is also obtained
from the pyrolysis gasoline stream in a naphtha steam cracker and by toluene disproportion.
[0010] Cost-effective methods for generating renewable PTA have not yet been developed to
date. PTA, toluene and other aromatic precursors are naturally degraded by some bacteria.
However, these degradation pathways typically involve monooxygenases that operate
irreversibly in the degradative direction. Hence, biosynthetic pathways for PTA are
severely limited by the properties of known enzymes to date.
[0011] A promising precursor for PTA is
p-toluate, also known as
p-methylbenzoate.
P-Toluate is an intermediate in some industrial processes for the oxidation of
p-xylene to PTA. It is also an intermediate for polymer stabilizers, pesticides, light
sensitive compounds, animal feed supplements and other organic chemicals. Only slightly
soluble in aqueous solution,
p-toluate is a solid at physiological temperatures, with a melting point of 275°C.
Microbial catalysts for synthesizing this compound from sugar feedstocks have not
been described to date.
[0012] Thus, there exists a need for alternative methods for effectively producing commercial
quantities of compounds such as styrene, 2,4-pentadienoate, 1,3-butadiene, p-toluate,
terephthalate, benzene and toluene. The present invention satisfies this need and
provides related advantages as well.
SUMMARY OF THE INVENTION
[0013] The invention provides non-naturally occurring microbial organisms having a toluene,
benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate,
benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol or 1,3-butadiene pathway. The invention
additionally provides methods of using such organisms to produce toluene, benzene,
p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate,
benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol or 1,3-butadiene.
[0014] The invention also provides non-naturally occurring microbial Organisms having a
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate (2H3M4OP) pathway, a p- toluate pathway,
a terephthalate pathway, a (2 -hydroxy-4-oxobutoxy)phosphonate (2H4OP) pathway, and/or
a benzoate pathway. The invention additionally provides methods of using such organisms
to produce (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, p- toluate, terephthalate,
(2-hydroxy-4-oxobutoxy)phosphonate, or benzoate.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015]
Figure 1 shows the conversion of phenylalanine to toluene via phenylacetate. Enzymes
are A. phenylalanine aminotransferase and/or phenylalanine oxidoreductase (deaminating),
B. phenylpyruvate decarboxylase, C. phenylacetaldehyde dehydrogenase and/or oxidase,
D. phenylacetate decarboxylase, E. phenylacetaldehyde decarbonylase, and F. phenylpyruvate
oxidase.
Figure 2 shows the conversion of phenylalalanine to benzene by phenylalanine benzene-lyase.
Figure 3 shows pathways to styrene from benzoyl-CoA. Enzymes are: A. benzoyl-CoA acetyltransferase,
B. 3-oxo-3-phenylpropionyl-CoA synthetase, transferase and/or hydrolase, C. benzoyl-acetate
decarboxylase, D. acetophenone reductase and E. 1-phenylethanol dehydratase, F. phosphotrans-3-oxo-3-phenylpropionylase,
G. benzoyl-acetate kinase.
Figure 4 shows the conversion of muconate stereoisomers to 1,3-butadiene. Enzymes
are A. trans, trans-muconate decarboxylase, B. cis, trans-muconate cis-decarboxylase, C. cis, trans-muconate trans-decarboxylase, D. cis, cis-muconate decarboxylase, E. trans-2,4-pentadienoate decarboxylase, F. cis-2,4-pentadienoate decarboxylase.
Figure 5 shows a schematic depiction of an exemplary pathway to (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate
(2H3M4OP) from glyceraldehyde-3-phosphate and pyruvate. G3P is glyceraldehyde-3-phosphate,
DXP is 1-deoxy-D-xylulose-5-phosphate and 2ME4P is C-mehyl-D-erythritol-4-phosphate.
Enzymes are (A) DXP synthase; (B) DXP reductoisomerase; and (C) 2ME4P dehydratase.
Figure 6 shows a schematic depiction of an exemplary alternate shikimate pathway to
p-toluate. Enzymes are: (A) 2-dehydro-3-deoxyphosphoheptonate synthase; (B) 3-dehydroquinate
synthase; (C) 3-dehydroquinate dehydratase; (D) shikimate dehydrogenase; (E) Shikimate
kinase; (F) 3-phosphoshikimate-2-carboxyvinyltransferase; (G) chorismate synthase;
and (H) chorismate lyase. Compounds are: (1) (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate;
(2) 2,4-dihydroxy-5-methyl-6-[(phosphonooxy)methyl]oxane-2-carboxylate; (3) 1,3-dihydroxy-4-methyl-5-oxocyclohexane-1-carboxylate;
(4) 5-hydroxy-4-methyl-3-oxocyclohex-1-ene-1-carboxylate; (5) 3,5-dihydroxy-4-methylcyclohex-1-ene-1-carboxylate;
(6) 5-hydroxy-4-methyl-3-(phosphonooxy)cyclohex-1-ene-1-carboxylate; (7) 5-[(1-carboxyeth-1-en-1-yl)oxy]-4-methyl-3-(phosphonooxy)cyclohex-1-ene-1-carboxylate;
(8) 3-[(1-carboxyeth-1-en-1-yl)oxy]-4-methylcyclohexa-1,5-diene-1-carboxylate; and
(9) p-toluate.
Figure 7 shows an exemplary pathway for conversion of p-toluate to terephthalic acid (PTA). Reactions A, B and C are catalyzed by p-toluate methyl-monooxygenase reductase, 4-carboxybenzyl alcohol dehydrogenase and
4-carboxybenzyl aldehyde dehydrogenase, respectively. The compounds shown are (1)
p-toluic acid; (2) 4-carboxybenzyl alcohol; (3) 4-carboxybenzaldehyde and (4) terephthalic
acid.
Figure 8 shows an exemplary pathway to (2-hydroxy-4-oxobutoxy)phosphonate from erythrose-4-phosphate.
Enzymes are: A. erythrose-4-phosphate dehydratase, B. (2,4-dioxobutoxy)phosphonate
reductase. Compounds are: (1) erythrose-4-phosphate, (2) (2,4-dioxobutoxy)phosphonate
and (3) (2-hydroxy-4-oxobutoxy)phosphonate.
Figure 9 shows an alternate shikimate pathway from (2-hydroxy-4-oxobutoxy)phosphonate
to benzoate. Enzymes are: A. 2-dehydro-3-deoxyphosphoheptonate synthase, B. 3-dehydroquinate
synthase, C. 3-dehydroquinate dehydratase, D. shikimate dehydrogenase, E. shikimate
kinase, F. 3-phosphoshikimate-2-carboxyvinyltransferase, G. chorismate synthase, H.
chorismate lyase. Compounds are: 1. (2-hydroxy-4-oxobutoxy)phosphonate, 2.2,4-dihydroxy-6-[(phosphonooxy)methyl]oxane-2-carboxylate,
3. 1,3-dihydroxy-5-oxocyclohexane-1-carboxylate, 4. 5-hydroxy-3-oxocyclohex-1-ene-1-carboxylate,
5. 3,5-dihydroxycyclohex-1-ene-1-carboxylate, 6. 5-hydroxy-3-(phosphonooxy)cyclohex-1-ene-1-carboxylate,
7. 5-[(1-carboxyeth-1-en-1-yl)oxy]-3-(phosphonooxy)cyclohex-1-ene-1-carboxylate, 8.
3-[(1-carboxyeth-1-en-1-yl)oxy]cyclohexa-1,5-diene-1-carboxylate, 9. benzoate.
Figure 10 shows pathways from benzoate and benzoyl-CoA to benzene. Enzymes are A.
benzoyl-CoA synthetase, transferase and/or hydrolase, B. benzoate reductase, C. benzaldehyde
decarbonylase, D. benzoyl-CoA reductase, E. benzoate decarboxylase, F. phosphotransbenzoylase,
G. (benzoyloxy)phosphonate reductase (dephosphorylating), H. benzoate kinase.
Figure 11 shows pathways from p-toluate (also called p-toluic acid) and p-methylbenzoyl-CoA to toluene. Enzymes are A. p-methylbenzoyl-CoA synthetase, transferase and/or hydrolase, B. p-toluate reductase, C. p-methylbenzaldehyde decarbonylase, D. p-methylbenzoyl-CoA reductase, E. p-toluate decarboxylase, F. phosphotrans-p-methylbenzoylase, G. (p-methylbenzoyloxy)phosphonate reductase (dephosphorylating), H. p-toluate kinase.
Figure 12 shows pathways to 2,4-pentadienoate from pyruvate. Enzymes are A. 4-hydroxy-2-oxovalerate
aldolase, B. 4-hydroxy-2-oxovalerate dehydratase, C. 2-oxopentenoate reductase, D.
2-hydroxypentenoate dehydratase, E. 4-hydroxy-2-oxovalerate reductase, F. 2,4-dihydroxypentanoate
2-dehydratase, G. 4-hydroxypent-2-enoate dehydratase and H. 2,4-dihydroxypentanoate
4-dehydratase.
Figure 13 shows pathways from alanine and ornithine to 2,4-pentadienoate. Enzymes
are A. AKP thiolase, B. AKP deaminase, C. acetylacrylate reductase, D. 4-hydroxypent-2-enoate
dehydratase, E. AKP aminotransferase and/or dehydrogenase, F. 2-hydroxy-4-oxopentanoate
dehydratase, G. 2,4-dihydroxypentanoate 2-dehydratase, H. 2,4-dioxopentactoate 2-reductase,
I. 2-hydroxy-4-oxopentanoate reductase, J. AKP reductase, K. 2,4-dioxopentanoate 4-reductase,
L. 2-amino-4-hydroxypentanoate aminotransferase and/or dehydrogenase, M. ornithine
4,5-aminomutase, N. 2,4-diaminopentanoate 4-aminotransferase and/or 4-dehydrogenase.
AKP is 2-amino-4-oxopentanoate.
Figure 14 shows additional pathways from ornithine to 2,4-pentadienoate. Enzymes are
A. ornithine 2,3-aminomutase, B. 3,5-diaminopentanoate deaminase, C. 5-aminopent-2-enoate
deaminase, D. 3,5-diaminopentanoate aminotransferase and/or dehydrogenase, E. 3-amino-5-oxopentanoate
deaminase, F. 5-oxopent-2-enoate reductase, G. 5-hydroxypent-2-enoate dehydratase,
H. 5-aminopent-2-enoate aminotransferase and/or dehydrogenase, I. 3-amino-5-oxopentanoate
reductase, J. 3-amino-5-hydroxypentanoate deaminase.
Figure 15 shows pathways from 3-hydroxypropanoyl-CoA and/or acrylyl-CoA to 2,4-pentadienoate.
Enzymes are A. 3-hydroxypropanoyl-CoA acetyltransferase, B. 3-oxo-5-hydroxypentanoyl-CoA
reductase, C. 3,5-dihydroxypentanoyl-CoA dehydratase, D. 5-hydroxypent-2-enoyl-CoA
dehydratase, E. pent-2,4-dionoyl-CoA synthetase, transferase and/or hydrolase, F.
3-oxo-5-hydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, G. 3,5-didhydroxypentamoyl-CoA
synthetase, transferase and/or hydrolase, H. 5-hydroxypent-2-enoyl-CoA synthetase,
transferase and/or hydrolase, I. 3-oxo-5-hydroxypentanoate reductase, J. 3,5-dihydroxypentanoate
dehydratase, K. 3-hydroxypropanoyl-CoA dehydratase, L. 3-oxo-5-hydroxypentanoyl-CoA
dehydratase, M. acrylyl-CoA acetyltransferase, N. 3-oxopent-4-enoyl-CoA reductase,
O. 3-oxopent-4-enoyl-CoA synthetase; transferase, and/or hydrolase, P. 3-oxopent-4-enoate
reductase, Q. 5-hydroxypent-2-enoate dehydratase, R. 3-hydroxypent-4-enoyl-CoA dehydratase,
S. 3-hydroxypent-4-enoate dehydratase. 3-HP-CoA is 3-hydroxypropanoyl-CoA.
Figure 16 shows the formation of butadiene from 3-hydroxypent-4-enoate (3HP4) by 3-hydroxypent-4-enoate
decarboxylase.
Figure 17 shows the formation of butadiene from 3,5-dihydroxypentanoate by 3,5-dihydroxypentanoate
decarboxylase and 3-butene-1-ol dehydratase. Dehydration of 3-butene-1-ol to butadiene
can also occur via chemical catalysis.
Figure 18 shows the formation of the 3-hydroxypent-4-enoate (3HP4) intermediate from
2,4-pentadienoate via 2,4-pentadienoate hydratase.
Figure 19 shows pathways to butadiene, 3-hydroxypent-4-enoate (3HP4), 2,4-pentadienoate
and 3-butene-1-ol from 3-HP-CoA and/or acrylyl-CoA. Enzymes are A. 3-hydroxypropanoyl-CoA
acetyltransferase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase, C. 3,5-dihydroxypentanoyl-CoA
dehydratase, D. 5-hydroxypent-2-enoyl-CoA dehydratase, E. pent-2,4-dienoyl-CoA synthetase,
transferase and/or hydrolase, F. 3-oxo-5-hydroxypentanoyl-CoA synthetase, transferase
and/or hydrolase, G. 3,5-dihydroxypentanoyl-CoA synthetase, transferase and/or hydrolase,
H. 5-hydroxypent-2-enoyl-CoA synthetase, transferase and/or hydrolase, I. 3-oxo-5-hydroxypentanoate
reductase, J. 3,5-dihydroxypentanoate dehydratase, K. 3-hydroxypropanoyl-CoA dehydratase,
L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase, M. acrylyl-CoA acetyltransferase, N.
3-oxopent-4-enoyl-CoA reductase, O. 3-oxopent-4-enoyl-CoA synthetase, transferase
and/or hydrolase, P. 3-oxopent-4-enoate reductase, Q. 5-hydroxypent-2-enoate dehydratase,
R. 3-hydroxypent-4-enoyl-CoA dehydratase, S. 3-hydroxypent-4-enoate dehydratase, T.
3-hydroxypent-4-enoyl-CoA transferase, synthetase or hydrolase, U. 3,5-dihydroxypentanoate
decarboxylase, V. 5-hydroxypent-2-enoate decarboxylase, W. 3-butene-1-ol dehydratase
(or chemical conversion), X. 2,4-pentadiene decarboxylase, Y. 3-hydroxypent-4-enoate
decarboxylase. 3-HP-CoA is 3-hydroxypropanoyl-CoA.
Figure 20 shows pathways to 3-hydroxypent-4-enoate (3HP4), 2,4-pentadienoate and butadiene
from succinyl-CoA. Enzymes are A. succinyl-CoA:acetyl-CoA acyltransferase, B. 3-oxoadipyl-CoA
transferase, synthetase or hydrolase, C. 3-oxoadipate dehydrogenase, D. 2-fumarylacetate
decarboxylase, E. 3-oxopent-4-enoate reductase, F. 3-hydroxypent-4-enoate dehydratase,
G. 3-oxoadipyl-CoA reductase, H. 3-hydroxyadipyl-CoA transferase, synthetase or hydrolase,
I. 3-hydroxyadipate dehydrogenase, J. 3-hydroxyhex-4-enedioate decarboxylase, K. 3-oxoadipate
reductase, L. 2-fumarylacetate reductase, M. 3-hydroxypent-4-enoate decarboxylase,
N. 2,4-pentadienoate decarboxylase.
Figure 21 shows pathways to 3-butene-1-ol, butadiene and 2,4-pentadienoate from malonyl-CoA
and acetyl-CoA. Enzymes for transformation of the identified substrates to products
include: A. malonyl-CoA:acetyl-CoA acyltransferase, B. 3-oxoglutaryl-CoA reductase
(ketone-reducing), C. 3-hydroxyglutary-CoA reductase (aldehyde formint), D. 3-hydroxy-5-oxopentanoate
reductase, E. 3,5-dihydroxypentanoate dehydratase, F. 5-hydroxypent-2-enoate dehydratase,
G. 3-hydroxyglutaryl-CoA reductase (alcohol forming), H. 3-oxoglutaryl-CoA reductase
(aldehyde forming), I. 3,5-dioxopentanoate reductase (aldehyde reducing), J. 5-hydroxy-3-oxopentanoate
reductase, K. 3-oxoglutaryl-CoA reductase (CoA reducing and alcohol forming), L. 3,5-dioxopentanoate
reductase (ketone reducing), M. 3,5-dihydroxypentanoate decarboxylase, N. 5-hydroxypent-2-enoate
decarboxylase, O. 3-butene-1-ol dehydratase (or chemical conversion), P. 2,4-pentadiene
decarboxylase.
Figure 22 shows the reverse TCA cycle for fixation of CO2 on carbohydrates as substrates. The enzymatic transformations are carried out by
the enzymes as shown.
Figure 23 shows the pathway for the reverse TCA cycle coupled with carbon monoxide
dehydrogenase and hydrogenase for the conversion of syngas to acetyl-CoA.
Figure 24 shows Western blots of 10 micrograms ACS90 (lane 1), ACS91 (lane2), Mta98/99
(lanes 3 and 4) cell extracts with size standards (lane 5) and controls of M. themoacetica
CODH (Moth_1202/1203) or Mtr (Moth_1197) proteins (50, 150, 250, 350, 450, 500, 750,
900, and 1000 ng).
Figure 25 shows CO oxidation assay results. Cells (M. thermoacetica or E. coli with
the CODH/ACS operon; ACS90 or ACS91 or empty vector: pZA33S) were grown and extracts
prepared. Assays were performed at 55 °C at various times on the day the extracts
were prepared. Reduction of methylviologen was followed at 578 nm over a 120 sec time
course.
DETAILED DESCRIPTION OF THE INVENTION
[0016] The present invention is directed, in part, to the design and production of cells
and organisms having biosynthetic production capabilities for toluene, benzene, styrene,
2,4-pentadienoate and 1,3-butadiene. The routes to toluene and benzene, Figures 1
and 2, begin with the naturally occurring amino acid phenylalanine and, thus, most
organisms will be capable of serving as a host for the construction of a non-naturally
occurring organism for the production of toluene and benzene. Strategies for enhancing
phenylalanine production are known in the art (
Yakandawala et al., App. Microbiol. Biotech. 78:283-291 (2008);
Lee et al., US Patent 5,008,190).
[0018] This invention is also directed, in part, to non-naturally occurring microorganisms
that express genes encoding enzymes that catalyze 1,3-butadiene production, as shown
in Figure 4. In some embodiments, pathways for the production of muconate are derived
from central metabolic precursors. Muconate is a common degradation product of diverse
aromatic compounds in microbes. Several biocatalytic strategies for making
cis,cis-muconate have been developed. Engineered
E. coli strains producing muconate from glucose via shikimate pathway enzymes have been developed
in the Frost lab (
U.S. Patent 5,487,987 (1996);
Niu et al., Biotechnol Prog., 18:201-211 (2002)). These strains are able to produce 36.8 g/L of
cis,cis-muconate after 48 hours of culturing under fed-batch fermenter conditions (22% of
the maximum theoretical yield from glucose). Muconate has also been produced biocatalytically
from aromatic starting materials such as toluene, benzoic acid and catechol. Strains
producing muconate from benzoate achieved titers of 13.5 g/L and productivity of 5.5
g/L/hr (
Choi et al., J. Ferment. Bioeng. 84:70-76 (1997)). Muconate has also been generated from the effluents of a styrene monomer production
plant (
Wu et al., Enzyme and Microbiology Technology 35:598-604 (2004)).
[0019] This invention is also directed, in part, to non-naturally occurring microorganisms
that express genes encoding enzymes that catalyze 2,4-pentadienoate production, as
shown in Figures 12-15. Any of these pathways can feed into a further 1,3-butadiene
pathway by inclusion of the requisite 2,4-pentadienoate decarboxylase. Figure 12 shows
the overall conversion of pyruvate to 2,4-pentadienoate by three pathways, Figure
13 shows the overall conversion of ornithine or alanine to 2,4-pentadienoate via common
intermediate 2-amino-4-ketopentanoate (AKP). Figure 13 shows six routes to 2,4-pentadienoate
from AKP, three of which intercept intermediates shown in Figure 12. Figure 14 shows
four additional routes to 2,4-pentadienoate from ornithine. Figure 15 shows numerous
routes to 2,4-pentadienoate from 3-hydroxypropanoyl-COA (3-HP-CoA) and acryloyl-CoA.
[0020] The invention is also directed, in part, to non-naturally occurring microbial organisms
that express genes encoding enzymes that catalyze 1,3-butadiene production, as shown
in Figures 16-17 and 19-21. Figure16 shows the decarboxylative dehydration of 3-hydroxypent-4-enoate
(3HP4) to 1,3-butadiene, where 3HP4 is available via pathways shown in Figures 15
and 19. 3HP4, being important in its own right, is shown in Figure 18 via intermediate
2,4-pentadienoate via hydration, as well as the via the pathways of Figures 15, 19,
and 20. Likewise, Figure 17 shows the tandem decarboxylative dehydration and elimination
(further dehydration) of intermediate 3,5-dihydroxypentanoate, which is itself accessible
through, the pathways shown in Figure 15, 19, and 21.
[0021] Figure 19 shows pathways to 1,3-butadiene and 1,3-butadiene intermediates from 3-HP-CoA
and acrylyl-CoA. Figure 20 shows pathways to 1,3-butadiene and 1,3-butadiene intermediates
from succinyl-CoA. The requisite succinyl-CoA is a central metabolic intermediate,
the yield of which can be enhanced via a reductive TCA cycle as described further
herein. Finally, figure 21 shows pathways to 1,3-butadiene and 1,3-butadiene intermediates
from the condensation of malonyl-CoA and acetyl-CoA, the latter also benefitting from
increased throughput via reductive TCA pathways described herein.
[0022] The present invention is also directed to the design and production of cells and
organisms having biosynthetic production capabilities for
p-toluate, terephthalate, (2-oxobutoxy)phosphonate, benzoate, or benzene. The results
described herein indicate that metabolic pathways can be designed recombinantly engineered
to achieve the biosynthesis of
p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxybutoxy)phosphonate, toluene, (2-hydroxy-4-oxobutoxy)phosphonate,
benzoate, or benzene in
Escherichia coli and other cells or organisms. Biosynthetic production of
p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, toluene, (2-hydroxy-4-oxobutoxy)phosphonate,
benzoate, or benzene can be confirmed by construction of strains having the designed
metabolic genotype. These metabolically engineered cells or organisms also can be
subjected to adaptive evolution to further augment
p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, toluene, (2-hydroxy-4-oxobutoxy)phosphonate,
benzoate, or benzene biosynthesis, including under conditions approaching theoretical
maximum growth.
[0023] The shikimate biosynthesis pathway in
E.
coli converts erythrose-4-phosphate to chorismate, an important intermediate that leads
to the biosynthesis of many essential metabolites including 4-hydroxybenzoate. 4-Hydroxybenzoate
is structurally similar to
p-toluate, an industrial precursor of terephthalic acid, and benzene. As disclosed herein,
shikimate pathway enzymes are utilized to accept the alternate substrate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate
(2H3M4OP), and transform it to
p-toluate or toluene, or the alternate substrate (2-hydroxy-4-oxobutoxy)phosphonate
(2H4OP) and transform it to benzoate or benzene. In addition, a pathway is used to
synthesize the 2H3M4OP or 2H4OP precursor using enzymes from the non-mevalonate pathway
for isoprenoid biosynthesis.
[0024] Disclosed herein are strategies for engineering a microorganism to produce renewable
p-toluate, terephthalate (PTA), toluene, benzoate, or benzene from carbohydrate feedstocks.
In the toluene series, glyceraldehyde-3-phosphate (G3P) and pyruvate are converted
to 2-hydroxy-3-methyl-4-oxobutoxy)phosphonate (2H3M4OP) in three enzymatic steps (see
Example III and Figure 5). The 2H3M4OP intermediate is subsequently transformed to
p-toluate by enzymes in the shikimate pathway (see Example IV and Figure 6).
p-Toluate can be further converted to PTA by a microorganism (see Example V and Figure
7). In the benzene series, 2H4OP is prepared by dehydration and reduction of erythrose-4-phosphate
(see Example VI and Figure 8). The 2H4OP intermediate is subsequently transformed
to benzoate by enzymes in the shikimate pathway (see Example VI and Figure 9). Benzoate
and
p-toluate are converted to benzene and toluene, respectively (see Example VII, and
Figures 10 and 11).
[0025] The conversion of G3P to
p-toluate requires one ATP, two reducing equivalents (NAD(P)H), and two molecules of
phosphoenolpyruvate, according to net reaction below.
G3P + 2 PEP + ATP +2 NAD(P)H + 2 H
+ →
p-Toluate + 4 Pi + ADP + 2 NAD(P)
+ + CO
2 + H
2O
[0026] An additional ATP is required to synthesize G3P from glucose. The maximum theoretical
p-toluate yield is 0.67 mol/mol (0.51 g/g) from glucose minus carbon required for energy.
Under the assumption that 2 ATPs are consumed per
p-toluate molecule synthesized, the predicted
p-toluate yield from glucose is 0.62 mol/mol (0.46 g/g)
p-totuate.
[0027] If
p-toluate is further converted to PTA by enzymes as described in Example III, the predicted
PTA yield from glucose is 0.64 mol/mol (0.58 g/g). In this case, the oxidation of
p-toluate to PTA generates an additional net reducing equivalent according to the net
reaction:
p-toluate + O
2 + NAD
+ → PTA + NADH + 2 H
+
[0028] Enzyme candidates for catalyzing each step of the above pathways are described in
the following sections. Successfully engineering pathways for the production of toluene,
benzene, styrene,
p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate,
benzoate or 1,3-butadiene entails identifying an appropriate set of enzymes with sufficient
activity and specificity, cloning their corresponding genes into a production host,
optimizing the expression of these genes in the production host, optimizing fermentation
conditions, and assaying for product formation following fermentation.
[0029] As used herein, the term "non-naturally occurring" when used in reference to a microbial
organism or microorganism of the invention is intended to mean that the microbial
organism has at least one genetic alteration not normally found in a naturally occurring
strain of the referenced species, including wild-type strains of the referenced species.
Genetic alterations include, for example, modifications introducing expressible nucleic
acids encoding metabolic polypeptides, other nucleic acid additions, nucleic acid
deletions and/or other functional disruption of the microbial organism's genetic material.
Such modifications include, for example, coding regions and functional fragments thereof,
for heterologous, homologous or both heterologous and homologous polypeptides for
the referenced species. Additional modifications include, for example, non-coding
regulatory regions in which, the modifications alter expression of a gene or operon.
Exemplary metabolic polypeptides include enzymes or proteins within a toluene, benzene,
p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, toluene, (2-hydroxy-4-oxobutoxy)phosphonate,
benzoate, styrene, or 1,3-butadiene biosynthetic pathway.
[0030] A metabolic modification refers to a biochemical reaction that is altered from its
naturally occurring state. Therefore, non-naturally occurring microorganisms can have
genetic modifications to nucleic acids encoding metabolic polypeptides, or functional
fragments thereof. Exemplary metabolic modifications are disclosed herein.
[0031] As used herein, the term "isolated" when used in reference to a microbial organism
is intended to mean an organism, that is substantially free of at least one component
as the referenced microbial organism is found in nature. The term includes a microbial
organism that is removed from some or all components as it is found in its natural
environment. The term also includes a microbial organism that is removed from some
or all components as the microbial organism is found in non-naturally occurring environments.
Therefore, an isolated microbial organism is partly or completely separated from other
substances as it is found in nature or as it is grown, stored or subsisted in non-naturally
occurring environments. Specific examples of isolated microbial organism include partially
pure microbes, substantially pure microbes and microbes cultured in a medium that
is non-naturally occurring.
[0032] As used herein, the terms "microbial," "microbial Organism" or "microorganism" are
intended to mean any organism that exists as a microscopic cell that is included within
the domains of archaea, bacteria or eukarya. Therefore, the term is intended to encompass
prokaryotic or eukaryotic cells or organisms having a microscopic size and includes
bacteria, archaea and eubacteria of all species as well as eukaryotic microorganisms
such as yeast and fungi. The term also includes cell cultures of any species that
can be cultured for the production of a biochemical.
[0033] As used herein, the term "CoA" or "coenzyme A" is intended to mean an organic cofactor
or prosthetic group (nonprotein portion of an enzyme) whose presence is required for
the activity of many enzymes (the apoenzyme) to form an active enzyme system. Coenzyme
A functions in certain condensing enzymes, acts in acetyl or other acyl group transfer
and in fatty acid synthesis and oxidation, pyruvate oxidation and in other acetylation.
[0034] As used herein, the term "(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate," abbreviated
herein as 2H3M4OP, has the chemical formula as shown in Figure 5. Such a compound
can also be described as 3-hydroxy-2-methyl butanal-4-phosphate.
[0035] As used herein, the term "(2-hydroxy-4-oxobutoxy)phosphonate," abbreviated herein
as 2H4OP, has the chemical formula as shown in Figure 8 (compound 3). Such a compound
can also be described as 3-hydroxybutanal-4-phosphate.
[0036] As used herein, the term "
p-toluate," having the molecular formula C
8H
7O
2- (see Figure 6, compound 9)(IUPAC name 4-methylbenzoate) is the ionized form of
p-toluic acid, and it is understood that
p-toluate and
p-toluic acid can be used interchangeably throughout to refer to the compound in any
of its neutral or ionized forms, including any salt forms thereof. It is understood
by those skilled in the art that the specific form will depend on the pH.
[0037] As used herein, the term "benzoate," having the molecular formula C
7H
6O
2 (see Figure 9, compound 9) is the ionized form of benzoic acid, and it is understood
that benzoate and benzoic acid can be used interchangeably throughout to refer to
the compound in any of it neutral or ionized forms, including any salt forms thereof.
It is understood by those skilled in the art that the specific form will depend on
the pH.
[0038] As used herein, the term "terephthalate," having the molecular formula C
8H
4O
4-2 (see Figure 7, compound 4)(IUPAC name terephthalate) is the ionized form of terephthalic
acid, also referred to as
p-phthalic acid or PTA, and it is understood that terephthalate and terephthalic acid
can be used interchangeably throughout to refer to the compound in any of its neutral
or ionized forms, including any salt forms thereof. It is understood by those skilled
understand that the specific form will depend on the pH.
[0039] As used herein, the term "substantially anaerobic" when used in reference to a culture
or growth condition is intended to mean that the amount of oxygen is less than about
10% of saturation for dissolved oxygen in liquid media. The term also is intended
to include sealed chambers of liquid or solid medium maintained with an atmosphere
of less than about 1% oxygen.
[0040] "Exogenous" as it is used herein is intended to mean that the referenced molecule
or the referenced activity is introduced into the host microbial organism. The molecule
can be introduced, for example, by introduction of an encoding nucleic acid into the
host genetic material such as by integration into a host chromosome or as non-chromosomal
genetic material such as a plasmid. Therefore, the term as it is used in reference
to expression of an encoding nucleic acid refers to introduction of the encoding nucleic
acid in an expressible form into the microbial organism. When used in reference to
a biosynthetic activity, the term refers to an activity that is introduced into the
host reference organism. The source can be, for example, a homologous or heterologous
encoding nucleic acid that expresses the referenced activity following introduction
into the host microbial organism. Therefore, the term "endogenous" refers to a referenced
molecule or activity that is present in the host. Similarly, the term when used in
reference to expression of an encoding nucleic acid refers to expression of an encoding
nucleic acid contained within the microbial organism. The term "heterologous" refers
to a molecule or activity derived from a source other than the referenced species
whereas "homologous" refers to a molecule or activity derived from the host microbial
organism. Accordingly, exogenous expression of an encoding nucleic acid of the invention
can utilize either or both a heterologous or homologous encoding nucleic acid.
[0041] It is understood that when more than one exogenous nucleic acid is included in a
microbial organism that the more than one exogenous nucleic acids refers to the referenced
encoding nucleic acid or biosynthetic activity, as discussed above. It is further
understood, as disclosed herein, that such more than one exogenous nucleic acids can
be introduced into the host microbial organism on separate nucleic acid molecules,
on polycistronic nucleic acid molecules, or a combination thereof, and still be considered
as more than one exogenous nucleic acid. For example, as disclosed herein, a microbial
organism can be engineered to express two or more exogenous nucleic acids encoding
a desired pathway enzyme or protein. In the case where two exogenous nucleic acids
encoding a desired activity are introduced into a host microbial organism, it is understood
that the two exogenous nucleic acids can be introduced as a single nucleic acid, for
example, on a single plasmid, on separate plasmids, can be integrated into the host
chromosome at a single site or multiple sites, and still be considered as two exogenous
nucleic acids. Similarly, it is understood that more than two exogenous nucleic acids
can be introduced into a host organism in any desired combination, for example, on
a single plasmid, on separate plasmids, can be integrated into the host chromosome
at a single site or multiple sites, and still be considered as two or more exogenous
nucleic acids, for example three exogenous nucleic acids. Thus, the number of referenced
exogenous nucleic acids or biosynthetic activities refers to the number of encoding
nucleic acids or the number of biosynthetic activities, not the number of separate
nucleic acids introduced into the host organism.
[0042] The non-naturally occurring microbial organisms of the invention can contain stable
genetic alterations, which refers to microorganisms that can be cultured for greater
than five generations without loss of the alteration. Generally, stable genetic alterations
include modifications that persist greater than 10 generations, particularly stable
modifications will persist more than about 25 generations, and more particularly,
stable genetic modifications will be greater than 50 generations, including indefinitely.
[0043] Those skilled in the art will understand that the genetic alterations, including
metabolic modifications exemplified herein, are described with reference to a suitable
host organism such as
E. coli and their corresponding metabolic reactions or a suitable source organism for desired
genetic material such as genes for a desired metabolic pathway. However, given the
complete genome sequencing of a wide variety of organisms and the high level of skill
in the area of genomics, those skilled in the art will readily be able to apply the
teachings and guidance provided herein to essentially all other organisms. For example,
the
E. coli metabolic alterations exemplified herein can readily be applied, to other species
by incorporating the same or analogous encoding nucleic acid from species other than
the referenced species. Such genetic alterations include, for example, genetic alterations
of species homologs, in general, and in particular, orthologs, paralogs or nonorthologous
gene displacements.
[0044] An ortholog is a gene or genes that are related by vertical descent and are responsible
for substantially the same or identical functions in different organisms. For example,
mouse epoxide hydrolase and human epoxide hydrolase can be considered orthologs for
the biological function of hydrolysis of epoxides. Genes are related by vertical descent
when, for example, they share sequence similarity of sufficient amount to indicate
they are homologous, or related by evolution from a common ancestor. Genes can also
be considered orthologs if they share three-dimensional structure but not necessarily
sequence similarity, of a sufficient amount to indicate that they have evolved from
a common ancestor to the extent that the primary sequence similarity is not identifiable.
Genes that are orthologous can encode proteins with sequence similarity of about 25%
to 100% amino acid sequence identity. Genes encoding proteins sharing an amino acid
similarity less that 25% can also be considered to have arisen by vertical descent
if their three-dimensional structure also shows similarities. Members of the serine
protease family of enzymes, including tissue plasminogen activator and elastase, are
considered to have arisen by vertical descent from a common ancestor.
[0045] Orthologs include genes or their encoded gene products that through, for example,
evolution, have diverged in structure or overall activity. For example, where one
species encodes a gene product exhibiting two functions and where such functions have
been separated into distinct genes in a second species, the three genes and their
corresponding products are considered to be orthologs. For the production of a biochemical
product, those skilled in the art will understand that the orthologous gene harboring
the metabolic activity to be introduced or disrupted is to be chosen for construction
of the non-naturally occurring microorganism. An example of orthologs exhibiting separable
activities is where distinct activities have been separated into distinct gene products
between two or more species or within a single species. A specific example is the
separation of elastase proteolysis and plasminogen proteolysis, two types of serine
protease activity, into distinct molecules as plasminogen activator and elastase.
A second example is the separation of mycoplasma 5'-3' exonuclease and
Drosophila DNA polymerase III activity. The DNA polymerase from the first species can be considered
an ortholog to either or both of the exonuclease or the polymerase from the second
species and vice versa.
[0046] In contrast, paralogs are homologs related by, for example, duplication followed
by evolutionary divergence and have similar or common, but not identical functions.
Paralogs can originate or derive from, for example, the same species or from a different
species. For example, microsomal epoxide hydrolase (epoxide hydrolase I) and soluble
epoxide hydrolase (epoxide hydrolase II) can be considered paralogs because they represent
two distinct enzymes, co-evolved from a common ancestor, that catalyze distinct reactions
and have distinct functions in the same species. Paralogs are proteins from the same
species with significant sequence similarity to each other suggesting that they are
homologous, or related through co-evolution from a common ancestor. Groups of paralogous
protein families include HipA homologs, luciferase genes, peptidases, and others.
[0047] A nonorthologous gene displacement is a nonorthologous gene from one species that
can substitute for a referenced gene function in a different species. Substitution
includes, for example, being able to perform substantially the same or a similar function
in the species of origin compared to the referenced function in the different species.
Although generally, a nonorthologous gene displacement will be identifiable as structurally
related to a known gene encoding the referenced function, less structurally related
but functionally similar genes and their corresponding gene products nevertheless
will still fall within the meaning of the term as it is used herein. Functional similarity
requires, for example, at least some structural similarity in the active site or binding
region of a nonorthologous gene product compared to a gene encoding the function sought
to be substituted. Therefore, a nonorthologous gene includes, for example, a paralog
or an unrelated gene.
[0048] Therefore, in identifying and constructing the non-naturally occurring microbial
organisms of the invention having toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate,
(2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol
or 1,3-butadiene biosynthetic capability, those skilled in the art will understand
with applying the teaching and guidance provided herein to a particular species that
the identification of metabolic modifications can include identification and inclusion
or inactivation of orthologs. To the extent that paralogs and/or nonorthologous gene
displacements are present in the referenced microorganism that encode an enzyme catalyzing
a similar or substantially similar metabolic reaction, those skilled in the art also
can utilize these evolutionally related genes.
[0049] Orthologs, paralogs and nonorthologous gene displacements can be determined by methods
well known to those skilled in the art. For example, inspection of nucleic acid or
amino acid sequences for two polypeptides will reveal sequence identity and similarities
between the compared sequences. Based on such similarities, one skilled in the art
can determine if the similarity is sufficiently high to indicate the proteins are
related through evolution from a common ancestor. Algorithms well known to those skilled
in the art, such as Align, BLAST, Clustal W and others compare and determine a raw
sequence similarity or identity, and also determine the presence or significance of
gaps in the sequence which can be assigned a weight or score. Such algorithms also
are known in the art and are similarly applicable for determining nucleotide sequence
similarity or identity. Parameters for sufficient similarity to determine relatedness
are computed based on well known methods for calculating statistical similarity, or
the chance of finding a similar match in a random polypeptide, and the significance
of the match determined. A computer comparison of two or more sequences can, if desired,
also be optimized visually by those skilled in the art. Related gene products or proteins
can be expected to have a high similarity, for example, 25% to 100% sequence identity.
Proteins that are unrelated can have an identity which is essentially the same as
would be expected to occur by chance, if a database of sufficient size is scanned
(about 5%). Sequences between 5% and 24% may or may not represent sufficient homology
to conclude that the compared sequences are related. Additional statistical analysis
to determine the significance of such matches given the size of the data set can be
carried out to determine the relevance of these sequences.
[0050] Exemplary parameters for determining relatedness of two or more sequences using the
BLAST algorithm, for example, can be as set forth below. Briefly, amino acid sequence
alignments can be performed using BLASTP version 2.0.8 (Jan-05-1999) and the following
parameters: Matrix: 0 BLOSUM62; gap open: 11; gap extension: 1; x_dropoff: 50; expect:
10.0; wordsize: 3; filter: on. Nucleic acid sequence alignments can be performed using
BLASTN version 2.0.6 (Sept-16-1998) and the following parameters: Match: 1; mismatch:
- 2; gap open: 5; gap extension: 2; x_dropoff: 50; expect: 10.0; wordsize: 11; filter:
off. Those skilled in the art will know what modifications can be made to the above
parameters to either increase or decrease the stringency of the comparison, for example,
and determine the relatedness of two or more sequences.
[0051] In some embodiment, the present invention provides a non-naturally occurring microbial
organism that includes a microbial organism having a toluene pathway which includes
at least one exogenous nucleic acid encoding a toluene pathway enzyme expressed in
a sufficient amount to produce toluene. The toluene pathway is selected from (A) 1)
one or both of phenylalanine aminotransferase and phenylalanine oxidoreductase (deaminating),
2) phenylpyruvate decarboxylase, and 3) phenylacetaldehyde decarbonylase; (B) 1) one
or more of phenylalanine aminotransferase and phenylalanine oxidoreductase (deaminating),
2) phenylpyruvate decarboxylase, 3) one or more of phenylacetaldehyde dehydrogenase
and phenylacetaldehyde oxidase, and 4) phenylacetate decarboxylase; and (C) one or
more of phenylalanine aminotransferase and phenylalanine oxidoreductase (deaminating),
2) phenylpyruvate oxidase, and 3) phenylacetate decarboxylase, as shown in the alternate
pathways in Figure 1.
[0052] The non-naturally occurring microbial organism having the toluene pathway can include
two exogenous nucleic acids each encoding a toluene pathway enzyme, three exogenous
nucleic acids each encoding a toluene pathway enzyme, four exogenous nucleic acids
each encoding a toluene pathway enzyme, or five exogenous nucleic acids each encoding
a toluene pathway enzyme. An exemplary non-naturally occurring microbial organism
having three exogenous nucleic acids can include an organism having genes encoding
1) phenylalanine aminotransferase and/or oxidoreductase (deaminating), 3) phenylpyruvate
oxidase, and 5) phenylacetate decarboxylase. An exemplary non-naturally occurring
organism having four exogenous nucleic acids can include an organism having exogenous
genes encoding 1) phonylalanine aminotransferase, 2) phenylalanine oxidoreductase
(deaminating), 3) phenylpyruvate decarboxylase, and 4) phenylacetaldehyde decarbonylase.
An exemplary non-naturally occurring microbial organism having five exogenous nucleic
acids can include an organism having genes encoding 1) phenylalanine aminotransferase,
2) phenylalanine oxidoreductase (deaminating), 3) phenylpyruvate decarboxylase, 4)
phenylacetaldehyde dehydrogenase and/or oxidase, and 5) phenylacetate decarboxylase.
Thus, in particular embodiments, a non-naturally occurring microbial organism can
have a complete toluene pathway every gene encoding every enzyme in a complete toluene
pathway. In some embodiments the non-naturally occurring microbial organism having
a toluene pathway can include at least one exogenous nucleic acid that is a heterologous
nucleic acid. Finally, the non-naturally occurring microbial organism having a toluene
pathway can be in a substantially anaerobic culture medium.
[0053] In some embodiments, the present invention provides a non-naturally occurring microbial
organism that includes a microbial organism having a benzene pathway which includes
at least one exogenous nucleic acid encoding a benzene pathway enzyme expressed in
a sufficient amount to produce benzene. The benzene pathway can include a phenylalanine
benzene-lyase as shown in Figures 2. The at least one exogenous nucleic acid can be
phenylalanine benzene-lyase itself or a nucleic acid that affects the production of
its precursor phenylalanine. In some embodiments the non-naturally occurring microbial
organism having a benzene pathway has at least one exogenous nucleic acid that is
a heterologous nucleic acid. In some embodiments, the non-naturally occurring microbial
organism having a benzene pathway is in a substantially anaerobic culture medium.
[0054] In some embodiments, the present invention provides a non-naturally occurring microbial
organism that includes a microbial organism having a styrene pathway which includes
at least one exogenous nucleic acid encoding a styrene pathway enzyme expressed in
a sufficient amount to produce styrene. The styrene pathway can be selected from (A)
1) benzoyl-CoA acetyltransferase, 2) one or more of 3-oxo-3-phenylpropionyl-CoA synthetase,
transferase, and hydrolase, 3) benzoyt-acetate decarboxylase, 4) acetopheone reductase,
and 5) 1-phenylethanol dehydratase; and (B) 1) benzoyl-CoA acetyltransferase, 2) phosphotrans-3-oxo-3-phenylpropionylase,
3) benzoyl-acetate kinase, 4) benzoyl-acetate decarboxylase, 5) acetopheone reductase,
and 6) 1-phenylethanol dehydratase, as indicated by the alternate pathways in Figure
3.
[0055] In some embodiments, the non-naturally occurring microbial organism having a styrene
pathway can include two exogenous nucleic acids each encoding a styrene pathway enzyme,
three exogenous nucleic acids each encoding a styrene pathway enzyme, four exogenous
nucleic acids each encoding a styrene pathway enzyme, five exogenous nucleic acids
each encoding a styrene pathway enzyme, six exogenous nucleic acids each encoding
a styrene pathway enzyme, and so on. An exemplary non-naturally occurring organism
having five exogenous nucleic acids can include an organism having exogenous genes
encoding 1) benzoyl-CoA acetyltransferase, 2) one of 3-oxo-3-phenylpropionyl-CoA synthetase,
transferase, and hydrolase, 3) benzoyl-acetate decarboxylase, 4) acetophenone reductase,
and 5) 1-phenylethanol dehydratase. An exemplary non-naturally occurring organism
having six exogenous nucleic acids can include an organism having exogenous genes
encoding 1) benzoyl-CoA acetyltransferase, 2) phosphotrans-3-oxo-3-phenylpropionylase,
3) benzoyl-acetate kinase, 4) benzoyl-acetate decarboxylase, 5) acetopheone reductase,
and 6) 1-phenylethanol dehydratase. In some embodiments the non-naturally occurring
microbial organism having a styrene pathway has at least one exogenous nucleic acid
that is a heterologous nucleic acid. In some embodiments the non-naturally occurring
microbial organism having a styrene pathway is in a substantially anaerobic culture
medium.
[0056] In some embodiments, the present invention provides a non-naturally occurring microbial
organism that includes a microbial organism having a 1,3-butadiene pathway which includes
at least one exogenous nucleic acid encoding a 1,3-butadiene pathway enzyme expressed
in a sufficient amount to produce 1,3-butadiene. The 1,3-butadiene pathway can be
selected from (A) 1)
trans, trans-muconate decarboxylase and 2)
trans-2,4-pentadienoate docarboxylase; (B) 1)
cis, trans-muconate
cis-decarboxylase and 2)
trans-2,4-pentadienoate decarboxylase; (C) 1)
cis,
trans-muconate
trans-decarboxylase 2)
cis-2,4-pentadienoate decarboxylase; and (D) 1)
cis,
cis-muconate decarboxylase and 2)
cis-2,4-pentadienoate decarboxylase, as indicated in the alternate pathways in Figure
4.
[0057] In some embodiments, the non-naturally occurring microbial organism having a 1,3-butadiene
pathway can include two exogenous nucleic acids each encoding a 1,3-butadiene pathway
enzyme. Thus, the two exogenous nucleic acids can encode a set selected from (A) 1)
trans, trans-muconate decarboxylase and 2)
trans-2,4-pentadienoate decarboxylase; (B) 1)
cis, trans-muconate
cis-decarboxylase and 2)
trans-2,4-pentadienoate decarboxylase; (C) 1)
cus, trans-muconate
trans-decarboxylase 2)
cis-2,4-pentadienoate decarboxylase; and (D) 1)
cis,
cis-muconate decarboxylase and 2)
cis-2,4-pentadienoate decarboxylase, corresponding to the complete pathways shown in
Figure 4. In some embodiments, the non-naturally occurring microbial organism having
a 1,3-butadiene pathway has at least one exogenous nucleic acid that is a heterologous
nucleic acid. In some embodiments, the non-naturally occurring microbial organism
having a 1,3-butadiene pathway is in a substantially anaerobic culture medium.
[0058] In some embodiments, the present invention provides a non-naturally occurring microbial
organism having a 2,4-pentadienoate pathway that includes at least one exogenous nucleic
acid encoding a 2,4-pentadienoate pathway enzyme expressed in a sufficient amount
to produce 2,4-pentadienoate. The 2,4-pentadienoate pathway has a set of enzymes capable
of converting AKP to 2,4-pentadienoate selected from (A) 1) a 4-hydroxy-2-oxovalerate
aldolase, 2) a 4-hydroxy-2-oxovalerate dehydratase, 3) a 2-oxopentenoate reductase,
and 4) a 2-hydroxypentenoate dehydratase, as shown in steps A-D of Figure 12, (B)
1) an AKP deaminase, 2) an acetylacrylate reductase, and 3) a 4-hydroxypent-2-enoate
dehydratase, as shown in steps B-D of Figure 13, (C) 1) an AKP aminotransferase and/or
dehydrogenase, 2) a 2,4-dioxopentanoate-2-reductase, 3) a 2-hydroxy-4-oxopentanoate
dehydratase, 4) an acetylacrylate reductase, and 5) a 4-hydroxypent-2-enoate dehydratase,
as shown in steps E, H, F, C, and D of Figure 13, (D) 1) an AKP aminotransferase and/or
dehydrogenase, 2) a 2,4-dioxopentanoate-4-reductase, 3) a 4-hydroxy-2-oxovalerate
dehydratase, 4) a 2-oxopentenoate reductase, and 5) a 2-hydroxypentenoate dehydratase,
as shown in steps E and K Figure 13, along with steps B-D of Figure 12, and (E) 1)
an AKP reductase, 2) a 2-amino-4-hydroxypentanoate aminotransferase and/or dehydrogenase,
3) a 4-bydroxy-2-oxovalerate dehydratase, 4) a 2-oxopentenoate reductase, and 5) a
2-hydroxypentenoate dehydratase, also shown in steps J and L of Figure 13, along with
steps B-D of Figure 12. In some embodiments, pathways of Figure 13 that include the
intermediate 4-hydroxy-2-oxovalerate, shown in Figure 12, can also be directed through
the 2,4-dihydroxypentanoate pathways shown in Figure 12 to provide 2,4-pentadienoate.
For example, from 4-hydroxy-2-oxovaterate, pathways to 2,4-pentadienoate can include
the enzymes in steps E, H, and D or steps E, F, and G, in Figure 12.
[0059] In some embodiments, the present invention also provides a non-naturally occurring
microbial organism having a 2,4-pentadienoate pathway that includes at least one exogenous
nucleic acid encoding a 2,4-pentadienoate pathway enzyme expressed in a sufficient
amount to produce 2,4-pentadienoate. The 2,4-pentadienoate pathway has a set of enzymes
selected from (A) 1) 4-hydroxy-2-oxovalerate aldolase, 2) 4-hydroxy-2-oxovaterate
reductase, 3) 2,4-dihydroxypentanoate 2-dehydratase, and 4) 4-hydroxypent-2-enoate
dehydratase, as shown in steps A, E, F, and G of Figure 12 and (B) 1) 4-hydroxy-2-oxovalerate
aldolase, 2) 4-hydroxy-2-oxovalerate reductase, 3) 2,4-dihydroxypentanoate 4-dehydratase
and 4) 2-hydroxypentenoate dehydratase, as shown in steps A, E, H, and D of Figure
12.
[0060] In some embodiments, the present invention also provides a non-naturally occurring
microbial organism having a 2,4-pentadienoate pathway that includes at least one exogenous
nucleic acid encoding a 2,4-pentadienoate pathway enzyme expressed in a sufficient
amount to produce 2,4-pentadienoate. The 2,4-pentadienoate pathway has a set of enzymes
selected from (A) 1) AKP aminotransferase and/or dehydrogenase, 2) 2,4-dioxopentanoate
2-reductase, 3) 2-hydroxy-4-oxopentanoate reductase, 4) 2,4-dihydroxypentanoate 2-dehydratase,
and 5) 4-hydroxypent-2-enoate dehydratase, as shown in steps E, H, I, G, and D of
Figure 13, and (B) 1) AKP aminotransferase and/or dehydrogenase, 2) 2,4-dioxopentanoate
2-reductase, 3) 2-hydroxy-4-oxopentanoate reductase, along with 4) 2,4-dihydroxypetanoate-2-dehydratase
and 5) 4-hydroxypent-2-enoate dehydratase or 4) 2,4-dihydroxypentanoate-4-dehydratase
and 5) 2-hydroxypentenoate dehydratase, as shown in steps E, H, and I of Figure 13,
along with steps F and G or H and D of Figure 12, respectively. That is to say, the
double dehydration of 2,4-dihydroxypentanoate can be performed in any order.
[0061] In some embodiments, the present invention also provides a non-naturally occurring
microbial organism having an AKP pathway that includes at least one exogenous nucleic
acid encoding an AKP pathway enzyme expressed in a sufficient amount to produce AKP.
The AKP pathway includes an ornithine 4,5-aminomutase and a 2,4-diaminopentanoate
4-aminotransferase and/or 4-dehydrogenase, as shown in steps M and N of Figure 13.
In some embodiments, the microbial organism having an AKP pathway includes two exogenous
enzymes encoding an ornithine 4,5-aminomutase and a 2,4-diaminopentanoate 4-aminotransferase
or 2,4-diaminopentanoate 4-dehydrogenase. In some embodiments, this AKP pathway can
be added to any of the aforementioned 2,4-pentadienoate pathways and as indicated
in Figure 13. Alternatively, AKP can be accessed from alanine by addition of an AKP
thiolase, as shown in step A of Figure 13, and fed into the various 2,4-pentadienoate
pathways described herein and shown in Figure 13, along with Figure 12.
[0062] In some embodiments, the present invention also provides a non-naturally occurring
microbial organism having a 2,4-pentadienoate pathway that includes at least one exogenous
nucleic acid encoding a 2,4-pentadienoate pathway enzyme expressed in a sufficient
amount to produce 2,4-pentadienoate. The 2,4-pentadienoate pathway has a set of enzymes
selected from (A) 1) ornithine 2,3-aminomutase, 2) 3,5-diaminopentanoate deaminase,
and 3) 5-aminopent-2-enoate deaminase, as shown in steps A-C of Figure 14, (B) 1)
ornithine 2,3-aminomutase, 2) 3,5-diaminopentanoate deaminase, 3) 5-aminopent-2-enoate
aminotransferase and/or dehydrogenase, 4) 5-oxopent-2-enoate reductase, and 5) 5-hydroxypent-2-enoate
dehydratase, as shown in steps A, B, H, F, and G of Figure 14, (C) 1) ornithine 2,3-aminomutase,
2) 3,5-diaminopentanoate aminotransferase and/or dehydrogenase, 3) 3-amino-5-oxopentanoate
deaminase, 4) 5-oxopent-2-enoate reductase, and 5) 5-hydroxypent-2-enoate dehydratase
as shown in steps A, D, E, F, and G of Figure 14, and (D) 1) ornithine 2,3-aminomutase,
2) 3,5-diaminopentanoate aminotransferase and/or dehydrogenase, 3) 3-amino-5-oxopentanoate
reductase, and 4) 3-amino-5-hydroxypentanoate deaminase, and 5) 5-hydroxypent-2-enoate
dehydratase as shown in steps A, D, I, J, and G of Figure 14.
[0063] In some embodiments, the present invention also provides a non-naturally occurring
microbial organism, having a 2,4-pentadienoate pathway that includes at least one
exogenous nucleic acid encoding a 2,4.-:pentadienoate pathway enzyme expressed, in
a sufficient amount to produce 2,4-pentadienoate. The 2,4-pentadienoate pathways has
a set of enzymes selected from any of the numerous pathways shown in Figure 15 starting
from 3-HP-CoA or acryloyl-CoA.
[0064] Exemplary pathways from 3-HP-CoA include the following enzyme sets (A) 1) 3-hydroxypropanoyl-CoA
acetyltransferase, 2) 3-oxo-5-hydroxypentanoyl-CoA reductase, 3) 3,5-dihydroxypentanoyl-CoA
dehydratase, 4) 5-hydroxypent-2-enoyl-CoA dehydratase, and 5) pent-2,4-dienoyl-CoA
synthetase, transferase and/or hydrolase, as shown in steps A-E of Figure 15, and
(B) 1) 3-hydroxypropanoyl-CoA acetyltransferase, 2) 3-oxy-5-hydroxypentanoyl-CoA synthetase,
transferase and/or hydrolase, 3) 3-oxo-5-hydroxypentanoate reductase, 4) 3,5-dihydroxypentanoate
dehydratase, and 5) 5-hydroxypent-2-enoate dehydratase, as shown in steps A, F, I,
J, and Q of Figure 15. One skilled in the art will recognize that enzyme sets defining
pathways (A) and (B) from 3-HP-CoA can be intermingled via reversible enzymes 3,5-hydroxypentanoyl-CoA
synthetase, transferase and/or hydrolase, as shown by step G in Figure 15, and 5-hydroxypent-2-enoyl-CoA
synthetase, transferase, and/or hydrolase, as shown by step H in Figure 15. Thus,
a 3-HP-CoA to 2,4-pentadienoate pathway can include the enzymes in steps A, B, G,
J, and Q, or steps A, B, C, H, and Q, or steps A, B, G, J, H, D, and E, or steps A,
F, I, G, C, D, and E, or steps, A, F, I, G, C, H, and Q, or steps A, F, I, J, H, D,
and E, each shown in Figure 15.
[0065] Exemplary pathway from acryloyl-CoA include the following enzyme sets (C) 1) acryloyl-CoA
acetyltransferase, 2) 3-oxopent-4-enoyl-CoA synthetase, transferase and/or hydrolase,
3) 3-oxopent-4-enoate reductase, 4) 3-hydroxypent-4-enoate dehydratase, as shown in
steps M, O, P, and S in Figure 15 and (D), 1) acryloyl-CoA acetyltransferase, 2) 3-oxopent-4-enoyl-CoA
reductase, 3) 3-hydroxypent-4-enoyl-CoA dehydratase, and 4) pent-2,4-dienoyl-CoA synthetase,
transferase, and/or hydrolase, as shown in steps M, N, R, and E. One skilled in the
art will recognize that enzyme sets defining pathway (A) and (B) from 3-HP-CoA and
(C) and (D) from acryloyl-CoA can be intermingled via reversible enzymes 3-hydroxypropanoyl-CoA
dehydratase, as shown in step K of Figure 15, and 3-oxo-5-hydroxypentanoyl-CoA dehydratase,
as shown in step L of Figure 15. Thus, step K can be added to any of the enumerated
pathways from acryloyl-CoA to 2,4-pentadienoate providing 2,4-pentadienoate pathways
such as steps K, M, N, R, and E or steps K, M, O, P, and S. Step K can also be used
a shuttle alternative to step A to provide 3-oxo-5-hydroxypentanoyl-CoA from 3-HP-CoA
via steps K, M, and L. Thus, any of the aforementioned pathways utilizing the enzyme
of step A can utilize the enzymes of steps K, M, and L, in its place. The save 3-oxo-5-hydroxypentanoyl-CoA
intermediate can be accessed from acryloyl-CoA by pathway via the enzymes of steps
K and A or M and L of Figure 15. Thus, acryloyl-CoA can be used to access all the
enumerated pathways that would be accessible from 3-HP-CoA. Thus, for example, an
acryloyl-CoA to 2,4-pentadienoate pathway can include enzymes from steps K, B, A,
B, C, D, and E, or steps K, A, F, I, J and Q, or steps K, A, B, G, J, and Q, or steps
K, A, B, G, J, H, D, and E, or steps K, A, B, C, H, and Q, or steps K, A, F, I, G,
C, D, and E, or steps K, A, F, I, G, C, H, Q, or steps K, A, F, I, J, H, D and E,
or steps M, L, B, C, D, and E, or steps M, L, F, I, J and Q, or steps M, L, B, G,
J, and Q, or steps M, L, B, G, J, H, D, and E, or steps M, L, B, C, H, and Q, or steps
M, L, F, I, G, C, D, and E, or steps M, L, F, I, G, C, H, Q, or steps M, L, F, I,
J, H, D and E, all as shown in Figure 15. Similarly, 3-HP-CoA can feed into the enumerated
acryloyl-CoA pathways via intermediate 3-oxopent-4-enoyl-CoA using the enzyme of step
L. Thus, a 3-HP-CoA to 2,4-pentadienoate pathway can include enzymes from steps A,
L, N, R, and E or steps A, L, O, P, and S, each pathway being shown in Figure 15.
[0066] In some embodiments, the present invention provides a non-naturally occurring microbial
organism, that includes a microbial organism having a 2,4-pentadienoate pathways which
includes at least one exogenous nucleic acid encoding a 2,4-pentadienoate pathway
enzyme expressed in a sufficient amount to produce 2,4-pentadienoate. The 2,4-pentadienoate
pathway has a set of enzymes selected from:
- 1) A. 3-hydroxypropanoyl-CoA acetyltransferase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase,
C. 3,5-dihydroxypentanoyt-CoA dehydratase, D. 5-hydroxypent-2-enoyl-CoA dehydratase,
E. pent-2,4-dienoyl-CoA synthetase, transferase and/or hydrolase;
- 2) A. 3-hydroxypropanoyl-CoA acetyltransferase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase,
G. 3,5-dihydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, J. 3,5-dihydroxypentanoate
dehydratase, H. 3-hydroxypent-2-enoyl-CoA synthetase, transferase, and/or hydrolase,
D. 5-hydroxypent-2-enoyl-CoA dehydratase, E. pent-2,4-dienoyl-CoA synthetase, transferase
and/or hydrolase;
- 3) A. 3-hydroxypropanoyl-CoA acetyltransferase, F. 3-oxo-5-hydroxypentanoyl-CoA synthetase,
transferase and/or hydrolase, I: 3-oxo-5-hydroxypentanoate reductase, G. 3,5-dihydroxypentanoyl-CoA
synthetase, transferase, and/or hydrolase, C. 3,5-dihydroxypentanoyl-CoA dehydratase,
D. 5-hydroxypent-2-enoyl-CoA dehydratase, E. pent-2,4-dienoyl-CoA synthetase, transferase
and/or hydrolase;
- 4) A. 3-hydroxypropanoyl-CoA acetyltransferase, F. 3-oxo-5-hydroxypentanoyl-CoA synthetase,
transferase and/or hydrolase, I. 3-oxo-5-hydroxypentanoate reductase, J. 3,5-dihydroxypentanoate
dehydratase, H. 5-hydroxypent-2-enoyl-CoA synthetase, transferase and/or hydrolase,
D. 5-hydroxypent-2-enoyl-CoA dehydratase, E. pent-2,4-dienoyl-CoA synthetase, transferase
and/or hydrolase;
- 5) K. 3-hydroxypropanoyl-CoA dehydratase, A. 3-hydroxypropanoyl-CoA acetyltransferase,
B. 3-oxo-5-hydroxypentanoyl-CoA reductase, C. 3,5-dihydroxypentanoyl-CoA dehydratase,
D. 5-hydroxypent-2-enoyl-CoA dehydratase, E. pent-2,4-dienoyl-CoA synthetase, transferase
and/or hydrolase;
- 6) K. 3-hydroxypropanoyl-CoA dehydratase, A. 3-hydroxypropanoyl-CoA acetyltransferase,
B. 3-oxo-5-hydroxypentanoyl-CoA reductase, G. 3,5-dihydroxypentanoyl-CoA synthetase,
transferase and/or hydrolase, J. 3,5-dihydroxypentanoate dehydratase, H. 5-hydroxypent-2-enoyl-CoA
synthetase, transferase and/or hydrolase, D. 5-hydroxypent-2-enoyl-CoA dehydratase,
E. pent-2,4-dienoyl-CoA synthetase, transferase and/or hydrolase,
- 7) K. 3-hydroxypropanoyl-CoA dehydratase, A. 3-hydroxypropanoyl-CoA acetyltransferase,
F. 3-oxo-5-hydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, I. 3-oxo-5-bydroxypentanoate
reductase, G. 3,5-dihydroxypentanoyl-CoA synthetase, transferase and/or hydrolase,
C. 3,5-dihydroxypentanoyl-CoA dehydratase, D. 5-hydroxypent-2-enoyl-CoA dehydratase,
E. pent-2,4-dienoyl-CoA synthetase, transferase and/or hydrolase;
- 8) K. 3-hydroxypropanoyl-CoA dehydratase, A. 3-hydroxypropanoyl-CoA acetyltransferase,
F. 3-oxo-5-hydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, I. 3-oxo-5-hydroxypentanoate
reductase, J. 3,5-dihydroxypentanoate dehydratase, H. hydroxypent-2-enoyl-CoA synthetase,
transferase and/or hydrolase, D. 5-hydroxypent-2-enoyl-CoA dehydratase, E. pent-2,4-dienoyl-CoA
synthetase, transferase and/or hydrolase;
- 9) M. acrylyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase,
B. 3-oxo-5-hydroxypetanoyl-CoA reductase, C. 3,5-dihydroxypentanoyl-CoA dehydratase,
D. 5-hydroxypent-2-enoyl-CoA dehydratase, E. pent-2,4-dienoyl-CoA synthetase, transferase
and/or hydrolase;
- 10) M. acrylyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase,
B. 3-oxo-5-hydroxypentanoyl-CoA reductase, G. 3,5-dihydroxypentanoyl-CoA synthetase,
transferase and/or hydrolase, J. 3,5-dihydroxypentanoate dehydratase, H. 5-hydroxypent-2-enoyl-CoA
synthetase, transferase and/or hydrolase, D. 5-hydroxypent-2-enoyl-CoA dehydratase,
E. pent-2,4-dienoyl-CoA synthetase, transferase and/or hydrolase;
- 11) M. acrylyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase,
3-hydroxypropanoyl-CoA acetyltransferase, F. 3-oxo-5-hydroxypentanoyl-CoA synthetase,
transferase and/or hydrolase, I: 3-oxo-5-hydroxypentanoate reductase, G. 3,5-dihydroxypentanoyl-CoA
synthetase, transferase and/or hydrolase, C. 3,5-dihydroxypentanoyl-CoA dehydratase,
D. 5-hydroxypent-2-enoyl-CoA dehydratase, E. pent-2,4-dienoyl-CoA synthetase, transferase
and/or hydrolase;
- 12) M. acrylyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl.-CoA dehydratase,
3-hydroxypropanoyl-CoA acetyltransferase, F. 3-oxo-5-hydroxypentanoyl-CoA synthetase,
transferase and/or hydrolase, I. 3-oxo-5-hydroxypentanoate reductase, J. 3,5-dihydroxypentanoate
dehydratase, H. 5-hydroxypent-2-enoyl-CoA synthetase, transferase and/or hydrolase,
D. 5-hydroxypent-2-enoyl-CoA dehydratase, E. pent-2,4-dienoyl-CoA synthetase, transferase
and/or hydrolase;
- 13) M. acrylyl-CoA acetyltransferase, N. 3-oxopent-4-enoyl-CoA reductase, R. 3-hydroxypent-4-enoyl-CoA
dehydratase, E. pent-2,4-dienoyl-CoA synthetase, transferase and/or hydrolase;
- 14) M. acrylyl-CoA acetyltransferase, N. 3-oxopent-4-enoyl-CoA reductase, T. 3-hydroxypent-4-enoyl-CoA
transferase, synthetase or hydrolase, S. 3-hydroxypent-4-enoate dehydratase; and
- 15) M. acrylyl-CoA. acetyltransferase, O. 3-oxopen-4-enoyl-CoA synthetase, transferase
and/or hydrolase, P. 3-oxopent-4-enoate reductase, S. 3-hydroxypent-4-enoate dehydratase;
- 16) A. 3-hydroxypropanoyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase,
N. 3-oxopent-4-enoyl-CoA reductase, R. 3-hydroxypent-4-enoyl-CoA dehydratase, E. pent-2,4-dienoyl-CoA
synthetase, transferase and/or hydrolase;
- 17) A. 3-hydroxypropanoyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase,
N. 3-oxopent-4-enoyl-CoA reductase, T. 3-hydroxypent-4-enoyl-CoA transferase, synthetase
or hydrolase, S. 3-hydroxypent-4-enoate dehydratase; and
- 18) A. 3-hydroxypropanoyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase,
O. 3-oxopent-4-enoyl-CoA synthetase, transferase and/or hydrolase, P. 3-oxopent-4-enoate
reductase, S. 3-hydroxypent-4-enoate dehydratase.
[0067] In some embodiments, the non-naturally occurring microbial organism of the invention
includes two, three, four, five, six, seven, or eight exogenous nucleic acids each
encoding a 2,4-pentadienoate pathway enzyme. In some embodiments, the non-naturally
occurring microbial organism of the invention has at least one exogenous nucleic acid
is a heterologous nucleic acid. In some embodiments, the non-naturally occurring microbial
organism of the invention is in a substantially anaerobic culture medium. In some
embodiments, the non-naturally occurring microbial organism of the invention further
includes a 2,4-pentadiene decarboxylase to convert 2,4-pentadienoate to 1,3-butadiene.
[0068] In some embodiments, a non-naturally occurring microbial organism includes a microbial
organism having a 1,3-butadiene pathway which includes at least one exogenous nucleic
acid encoding a 1,3-butadiene pathway enzyme expressed in a sufficient amount to produce
1,3-butadiene. The 1,3-butadiene pathways has a set of enzymes selected from:
- 1) M. acrylyl-CoA acetyltransferase, N. 3-oxopent-4-enoyl-CoA reductase, T. 3-hydroxypent-4-enoyl-CoA
transferase, synthetase or hydrolase, Y. 3-hydroxypent-4-enoate decarboxylase;
- 2) M. acrylyl-CoA acetyltransferase, O. 3-oxopent-4-enoyl-CoA synthetase, transferase
and/or hydrolase, P. 3-oxopent-4-enoate reductase, Y. 3-bydroxypent-4-enoate decarboxylase;
- 3) K. 3-hydroxypropanoyl-CoA dehydratase, M. acrylyl-CoA acetyltransferase, N. 3-oxopent-4-enoyl-CoA
reductase, T. 3-hydroxypent-4-enoyl-CoA tansferase, synthetase or hydrolase, Y. 3-hydroxypent-4-enoate
decarboxylase;
- 4) K. 3-hydroxypropanoyl-CoA dehydratase, M. acrylyl-CoA acetyltransferase, O. 3-oxopent-4-enoyl-CoA
synthetase, transferase and/or hydrolase, P. 3-oxopent-4-enoate reductase, Y. 3-hydroxypent-4-enoate
decarboxylase;
- 5) A. 3-hydroxypropanoyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase,
N. 3-oxopent-4-enoyl-CoA reductase, T. 3-hydroxypent-4-enoyl-CoA transferase, synthetase
or hydrolase, Y. 3-hydroxypent-4-enoate decarboxylase;
- 6) A. 3-hydroxypropanoyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase,
O. 3-oxopent-4-enoyl-CoA synthetase, transferase and/or hydrolase, P. 3-oxopent-4-enoate
reductase, Y. 3-hydroxypent-4-enoate decarboxylase,
[0069] In some embodiments, the non-naturally occurring microbial organism of the invention
includes two, three, four, or five exogenous nucleic acids each encoding a 1,3-butadiene
pathway enzyme. In some embodiments, the non-naturally occurring microbial organism
of the invention includes at least one exogenous nucleic acid that is a heterologous
nucleic acid. In some embodiments, the non-naturally occurring microbial organism
of the invention is in a substantially anaerobic culture medium.
[0070] In some embodiments, the present invention provides a non-naturally occurring microbial,
organism, that includes a microbial, organism having a 1,3-butadiene pathway which
includes at least one exogenous nucleic acid encoding a 3-butene-1-ol pathway enzyme
expressed in a sufficient amount to produce 3-butene-1-ol. The 3-butene-1-ol pathway
has a set of enzymes selected from:
- 1) A. 3-hydroxypropanoyl-CoA acetyltransferase, F. 3-oxo-5-hydroxypentanoyl-CoA synthetase,
transferase and/or hydrolase, I. 3-oxo-S-hydroxypentanoate reductase, U. 3,5-dihydroxypentanoate
decarboxylase;
- 2) A. 3-hydroxypropanoyl-CoA acetyltransferase, F. 3-oxo-5-hydroxypentanoyl-CoA synthetase,
transferase and/or hydrolase, I. 3-oxo-5-hydroxypentanoate reductase, J. 3,5-dihydroxypentanoate
dehydratase, V. 5-hydroxypent-2-enoate decarboxylase;
- 3) A. 3-hydroxypropanoyl-CoA acetyltransferase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase,
G. 3,5-dihydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, U. 3,5-dihydroxypentanoate
decarboxylase;
- 4) A. 3-hydroxypropanoyl-CoA acetyltransferase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase,
G. 3;5-dihydroxypentanoyl-CoA synthetase, transferase and/or hydrolas, J. 3,5-dihydroxypentanoate
dehydratase, V. 5-hydroxypent-2-enoate decarboxylase;
- 5) A. 3-hydroxypropanoyl-CoA acetyltransferase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase,
C. 3,5-dihydroxypentanoyl-CoA dehydratase, H. 5-hydroxypent-2-enoyl-CoA synthetase,
transferase and/or hydrolase, V. 5-hydroxypent-2-enoate docarboxylase;
- 6) M. acrylyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase,
F. 3-oxo-5-hydroxypentanoyl-CoOA synthetase, transferase and/or hydrolase, I. 3-oxy-5-hydroxypentanoate
reductase, U. 3,5-dihydroxypentanoate decarboxylase;
- 7) M. acrylyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase,
F. 3-oxo-5-hydroxypentanoyl-CoA synthetase, transferase and/or hydrotase, I. 3-oxo-5-hydroxypentanoate
reductase, J. 3,5-dihydroxypentanoate dehydratase, V. 5-hydroxypent-2-enoate decarboxylase;
- 8) M. acrylyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase,
B. 3-oxo-5-hydroxypentanoyl-CoA reductase, G. 3,5-dihydroxypentanoyl-CoA synthetase,
transferase and/or hydrolase, U. 3,5-dihydroxypentanoate decarboxylase;
- 9) M. acrylyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase,
B. 3-oxo-5-hydroxypentanoyl-CoA reductase, G. 3,5-dihydroxypentanoyl-CoA synthetase,
transferase, and/or hydrolase, J. 3,5-dihydroxypentanoate dehydratase, V. 5-hydroxypent-2-enoate
decarboxylase;
- 10) M. acrylyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase,
B. 3-oxo-5-hydroxypentanoyl-CoA reductase, C. 3,5-dihydroxypentanoyl-CoA dehydratase,
H. 5-hydroxypent-2-enoyl-CoA synthetase, transferase and/or hydrolase, V. 5-hydroxypent-2-enoate
decarboxylase;
- 11) K. 3-hydroxypropanoyl-CoA dehydratase, A. 3-hydroxypropanoyl-CoA acetyltransferase,
F. 3-oxo-5-hydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, I. 3-oxo-5-hydroxypentanoate
reductase, U. 3,5-dihydroxypentanoate decarboxylase;
- 12) K. 3-hydroxypropanoyl-CoA dehydratase, A. 3-hydroxypropanoyl-CoA acetyltransferase,
F. 3-oxo-5-hydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, I. 3-oxo-5-hydroxypentanoate
reductase, J. 3,5-dihydroxypentanoate dehydratase, V. 5-hydroxypent-2-enoate docarboxylase;
- 13) K. 3-hydroxypropanoyl-CoA dehydratase, A. 3-hydroxypropanoyl-CoA acetyltransferase,
B. 3-oxo-5-hydroxypentanoyl-CoA reductase, G. 3,5-dihydroxypentanoyl-CoA synthetase,
transferase and/or hydrolase, U. 3,5-dihydroxypentanoate decarboxylase;
- 14) K. 3-hydroxypropanoyl-CoA dehydratase, A. 3-hydroxypropanoyl-CoA acetyltransferase,
B. 3-oxo-5-hydroxypentanoyl-CoA reductase, G. 3,5-dihydroxypentanoyl-CoA synthetase,
transferase and/or hydrolase, J. 3,5-dihydroxypentanoate dehydratase, V. 5-hydroxypent-2-enoate
decarboxylase;
- 15) K. 3-hydroxypropanoyl-CoA dehydratase, A. 3-hydroxypropanoyl-CoA acetyltransferase,
B. 3-oxo-5-hydroxypentanoyl-CoA reductase, C. 3,5-dihydroxypentanoyl-CoA dehydratase,
H. 5-hydroxypent-2-enoyl-CoA synthetase, transferase and/or hydrolase, V. 5-hydroxypent-2-enoate
decarboxylase;
- 16) K. 3-hydroxypropanoyl-CoA dehydratase, M. acrylyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA
dehydratase, F. 3-oxo-5-hydroxypentanoyl-CoA synthetase, transferase and/or hydrolase,
1. 3-oxo-5-hydroxypentanoate reductase, U. 3,5-dihydroxypentanoate decarboxylase;
- 17) K. 3-hydroxypropanoyl-CoA dehydratase, M. acrylyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA
dehydratase, F. 3-oxo-5-hydroxypentanoyl-CoA synthetase, transferase and/or hydrolase,
I. 3-oxo-5-hydroxypentanoate reductase, J. 3,5-dihydroxypentanoate dehydratase, V.
5-hydroxypent-2-enoate decarboxylase;
- 18) K. 3-hydroxypropanoyl-CoA dehydratase, M. acrytyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA
dehydratase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase, G. 3,5-dihydroxypentanoyl-CoA
synthetase, transferase and/or bydrolase, U. 3,5-dihydroxypentanoate decarboxylase;
- 19) K. 3-hydroxypropanoyl-CoA dehydratase, M. acrylyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA
dehydratase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase, G. 3,5-dihydroxypentanoyl-CoA
synthetase, transferase and/or hydrolase, J. 3,5-dihydroxypentanoate dehydratase,
V. 5-hydroxypent-2-enoate decarboxylase;
- 20) K. 3-hydroxypropanoyl-CoA dehydratase, M. acrytyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA
dehydratase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase, C. 3,5-dihydroxypentanoyl-CoA
dehydratase, H. 5-hydroxypent-2-enoyl-CoA synthetase, transferase and/or hydrotase,
V. 5-hydroxypent-2-enoate decarboxylase;
[0071] In some embodiments, the non-naturally occurring microbial organism of the invention
includes two, three, four, five, six, or seven, exogenous nucleic acids each encoding
a 3-butene-1-ol pathway enzyme. In some embodiments, the non-naturally occurring microbial
organism of the invention has at least one exogenous nucleic acid that is a heterologous
nucleic acid. In some embodiments, the non-naturally occurring microbial organism
of the invention is in a substantially anaerobic culture medium. In some embodiments,
the non-naturally occurring microbial organism of the invention further includes a
3-butene-1-ol dehydratase to convert 3-butene-1-ol to 1,3-butadiene.
[0072] In some embodiments, non-naturally occurring microbial organism of the invention
can include two exogenous nucleic acids each encoding a 2,4-pentadienoate pathway
enzyme. In some embodiments, non-naturally occurring microbial organism of the invention
can include three exogenous nucleic acids each encoding a 2,4-pentadienoate pathway
enzyme. For example, the non-naturally occurring microbial organism of the invention
can include three exogenous nucleic encoding i) an AKP deaminase, ii) an acetylacrylate
reductase, and iii) a 4-hydroxypent-2-enoate dehydratase, thus providing an alanine
or ornithine accessible pathway to 2,4-pentadienoate via AKP. One skilled in the art
will recognize that this is merely exemplary and that three exogenous nucleic acids
can be the basis of any 2,4-pentadienoate-producing non-naturally occurring organism
in any of the enumerated pathway of Figure 12-15.
[0073] In some embodiments, the non-naturally occurring microbial organism of the invention
microbial can include any four exogenous nucleic acids each encoding a 2,4-pentadienoate
pathway enzyme. For example, a non-naturally occurring microbial organism can include
four exogenous nucleic acids encoding i) a 4-hydroxy-2-oxovalerate aldolase, ii) a.
4-hydroxy-2-oxovalerate dehydratase, iii) a 2-oxopentenoate reductase, and iv) a 2-hydroxypentenoate
dehydratase, thus defining a complete pathway from pyruvate to 2,4-pentadienoate,
as shown in Figure 12, One skilled in the art will recognize that this is merely exemplary
and that four exogenous nucleic acids can be the basis of any 2,4-pentadienoate-producing
non-naturally occurring organism in any of the enumerated pathways of Figure 12-15.
[0074] In still further embodiments, the non-naturally occurring microbial organism of the
invention can include five exogenous nucleic acids each encoding a 2,4-pentadienoate
pathway enzyme. Exemplary non-naturally occurring microbial organism of the invention
having five exogenous nucleic acids can include enzymes encoding (A) i) an AKP aminotransferase
and/or dehydrogenase, ii) a 2,4-dioxopentanoate-2-reductase, iii) a 2-hydroxy-4-oxopentanoate
dehydratase, iv) an acetylacrylate reductase, and v) a 4-hydroxypent-2-enoate dehydratase,
as shown in steps E, H, F, C, and D in Figure 13, or (B) i) an AKP aminotransferase
and/or dehydrogenase, ii) a 2,4-dioxopentanoate-4-reductase, iii) a 4-hydroxy-2-oxovalerate
dehydratase, iv) a 2-oxopentenoate reductase, and v) a 2-hydroxypentenoate dehydratase,
as shown in steps E and K of Figure 13, along with steps B, C, and D of Figure 12,
or i) an AKP reductase, ii) a 2-amino-4-hydroxypentanoate aminotransferase and/or
dehydrogenase, iii) a 4-hydroxy-2-oxovalerate dehydratase, iv) a 2-oxopentenoate reductase,
and v) a 2-hydroxypentenoate dehydratase, as shown in steps J and L of Figure 13,
along with steps B, C, and D of Figure 12. One skilled in the art will recognize that
this is merely exemplary and that five exogenous nucleic acids can be the basic of
any 2,4-pentadienoate-producing non-naturally occurring organism in any of the enumerated
pathway of Figure 12-15. Thus, in some embodiments two, three, four, five, six, up
to all of the enzymes in a 2,4-pentadienoate pathway can be provided insertion of
exogenous nucleic acids. In some embodiments, the non-naturally occurring microbial
organism of the invention has at least one exogenous nucleic acid is a heterologous
nucleic acid. Moreover, in some embodiments, the non-naturally occurring microbial
organism of the invention can be provided in a substantially anaerobic culture medium.
[0075] In some embodiments, the non-naturally occurring microbial organism of the invention
can further include a 2,4-pentadienoate decarboxylase expressed in a sufficient amount
to produce 1,3-butadiene by conversion of 2,4-pentadienoate to 1,3-butadiene. Thus,
any 2,4-pentadienoate pathway of Figure 12 can form the basis of further production
or 1,3 butadiene, as indicated by the conversion of cis or trans 2,4-pentadienoate
to 1,3-butadiene in Figure 4.
[0076] In some embodiments, the invention provides a non-naturally occurring microbial organism
having a toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate,
(2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol
or 1,3-butadiene pathway, wherein the non-naturally occurring microbial organism comprises
at least one exogenous nucleic acid encoding an enzyme or protein that converts a
substrate to a product. For example, in a toluene pathway (Figure 1), such substrate
to product is selected from the group consisting of phenylalanine to phenylpyruvate,
phenylpyruvate to phenylacetaldehyde, phenylpyruvate to phenylacetate, phenylacetaldehyde
to phenylacetate, phenylacetaldehyde to toluene, and phenylacetate to toluene. In
a styrene pathway (Figure 3), such a substrate to product is selected from the group
consisting of benzoyl-CoA to 3-oxo-3-phenylpropionyl-CoA, 3-oxy-3-phenylpropionyl-CoA
to [(3-oxo-3-phenylpropionyl)oxy] phosphonate, [(3-oxo-3-phenylpropionyl)oxy] phosphonate
to benzoyl-acetate, 3-oxo-3-phenylpropionyl-CoA to benzoyl-acetate, benzoyl-acetate
to acetophenone, acetophenone to 1-phenylethanol, and 1-phenylethanol to styrene.
In a 1,3-butadiene pathway (Figure 4), such substrate to product is selected from
trans, trans-muconate to
trans-2,4-pentadienoate,
cis,
trans-muconate to
trans-2,4-pentadienoate,
cis,trans-muconate to
cis-2,4-pentadienoate,
cis,cis-muconate to
cis-2,4-pentadienoate,
trans-2,4-pentadienoate to 1,3-butadiene, and
cis-2,4-pentadienoate to 1,3-butadiene. One skilled in the art will understand that these
are merely exemplary and that any of the substrate-product pairs disclosed herein
suitable to produce a desired product and for which an appropriate activity is available
for the conversion of the substrate to the product can be readily determined by one
skilled in the art based on the teachings herein.
[0077] In a 2,4-pentadienoate pathway, such a substrate to product is selected from pyruvate
to 4-hydroxy-2-oxovaterate, 4-bydroxy-2-oxovalerate to 2-oxopentenoate, 2-oxopentenoate
to 2-hydroxypentenoate, 2-hydroxypentenoate to 2,4-pentadienoate, AKP to acetylacrylate,
acetylacrylate to 4-hydroxypent-2-enoate, 4-hydroxypent-2-enoate to 2,4-pentadienoate,
AKP to 2,4-dioxopentanoate, 2,4-dioxopentanoate to 4-hydroxy-2-oxovalerate, AKP to
2-amino-4-hydroxypentanoate, 2-amino-4-hydroxypentanoate to 4-hydroxy-2-oxovalerate,
ornithine to 2,4-diaminopentanoate, 2,4-diaminopentanoate to AKP, alanine to AKP,
and so on.
[0078] The invention also provides a non-naturally occurring microbial organism, comprising
a microbial organism having a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway
comprising at least one exogenous nucleic acid encoding a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate
pathway enzyme expressed in a sufficient amount to produce (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate,
the (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway comprising 2-C-methyl-D-erythritol-4-phosphate
dehydratase (see Example III and Figure 5, step C). A non-naturally occurring microbial
organism comprising a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathways can further
comprise 1-deoxyxylulose-5-phosphate synthase or 1-deoxy-D-xylulose-5-phosphate reductoisomerase
(see Example III and Figure 5, steps A and B). Thus, a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate
can comprise 5 2-C-methyl-D-erythritol-4-phosphate dehydratase, 1-deoxyxylulose-5-phosphate
synthase and 1-deoxy-D-xylulose-5-phosphate reductoisomerase.
[0079] The invention also provides a non-naturally occurring microbial organism, comprising
a microbial organism having a
p-toluate pathway comprising at least one exogenous nucleic acid encoding a
p-toluate pathway enzyme expressed in a sufficient amount to produce
p-toluate, the
p-toluate pathway comprising 2-dehydro-3-deoxyphosphoheptonate synthase; 3-dehydroquinate
synthase; 3-dehydroquinate dehydratase; shikimate dehydrogenase; shikimate kinase;
3-phosphoshikimate-2-carboxyvinyltransferase; chorismate synthase; or chorismate lyase
(see Example IV and Figure 6, steps A-H). A non-naturally occurring microbial organism
having a
p-toluate pathway can further comprise a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate
pathway (Figure 5). A (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway can comprise,
for example, 2-C-methyl-D-erythritol-4-phosphate dehydratase, 1-deoxyxylulose-5-phosphate
synthase or 1-deoxy-D-xylulose-5-phosphate reductoisomerase (Figure 5).
[0080] The invention additionally provides a non-naturally occurring microbial organism,
comprising a microbial organism having a terephthalate pathway comprising at least
one exogenous nucleic acid encoding a terephthalate pathway enzyme expressed in a
sufficient amount to produce terephthalate, the terephthalate pathway comprising
p-toluate methyl-monooxygenase reductase; 4-carboxybenzyl alcohol dehydrogenase; or
4-carboxybenzyl aldehyde dehydrogenase (see Example V and Figure 7). Such an organism
containing a terephthalate pathway can additionally comprise a
p-toluate pathway, wherein thep
p-toluate pathway comprises 2-dehydro-3-deoxyphosphoheptonate synthase; 3-dehydroquinate
synthase; 3-dehydroquinate dehydratase; shikimate dehydrogenase; shikimate kinase;
3-phosphoshikimate-2-carboxyvinyltransferase; chorismate synthase; or chorismate lyase
(see Examples IV and V and Figures 6 and 7). Such a non-naturally occurring microbialorganism
having a terephthalate pathway and a
p-toluate pathway can further comprise a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate
pathway (see Example III and Figure 5). A (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate
pathway can comprise, for example, 2-C-methyl-D-erythritol-4-phosphate dehydratase,
1-deoxyxylulose-5-phosphate synthase or 1-deoxy-D-xylulose-5-phosphate reductoisomerase
(see Example III and Figure 5).
[0081] In some embodiments, the present invention provides a non-naturally occurring microbial
organism having a toluene pathway comprising at least one exogenous nucleic acid encoding
a toluene pathway enzyme expressed in a sufficient amount to produce toluene. The
toluene pathway is selected from a set of pathway enzymes selected from: a)
p-toluate decarboxylase; b)
p-toluate reductase and
p-methylbenzaldehyde decarbonylase; c)
p-toluate kinase, (
p-methylbenzoyloxy)phosphonate reductase, and
p-methylbenzaldehyde decarbonylase; d) (
p-methylbenzoyl-CoA synthetase, transferase and/or hydrolase), phosphotrans-
p-methylbenzoylase,
(p-methylbenzoyloxy)phosphonate reductase, and
p-methylbenzaldehyde decarbonylase; and e) (
p-methylbenzoyl-CoA synthetase, transferase and/or hydrolase),
p-methylbenzoyl-CoA reductase and
p-methylbenzaldehyde decarbonylase.
[0082] In some embodiments, the present invention provides a non-naturally occurring microbial
organism having a (2-hydroxy-4-oxobutoxy)phosphonate pathway comprising at least one
exogenous nucleic acid encoding a (2-hydroxy-4-oxobutoxy)phosphonate pathway enzyme
expressed in a sufficient amount to produce (2-hydroxy-4-oxobutoxy)phosphonate. The
(2-hydroxy-4-oxobutoxy)phosphonate pathway includes erythrose-4-phosphate dehydratase
and (2,4-dioxobutoxy) phosphonate reductase.
[0083] In some embodiments, the present invention provides a non-naturally occurring microbial
organism having a benzoate pathway comprising at least one exogenous nucleic acid
encoding a benzoate pathway enzyme expressed in a sufficient amount to produce benzoate.
The benzoate pathway includes 2-dehydro-3-deoxyphosphoheptonate synthase; 3-dehydroquinate
synthase; 3-dehydroquinate dehydratase; shikimate dehydrogenase; shikimate kinase;
3-phosphoshikimate-2-carboxyvinyltransferase; chorismate synthase; and chorismate
lyase.
[0084] In some embodiments, the present invention provides a non-naturally occurring microbial
organism having a benzene pathway comprising at least one exogenous nucleic acid encoding
a benzene pathway enzyme expressed in a sufficient amount to produce benzene. The
benzene pathway is selected from a set of pathway enzymes selected from: a) benzoate
decarboxylase; b) benzoate reductase and benzaldehyde decarbonylase; c) benzoate kinase,
(benzoyloxy)phosphonate reductase, and benzaldehyde decarbonylase; d) (benzoyl-CoA
synthetase, transferase and/or hydrolase), phosphotransbenzoylase, (benzoyloxy)phosphonate
reductase, and benzaldehyde decarbonylase; and e) (benzoyl-CoA synthetase, transferase
and/or hydrolase), benzoyl-CoA reductase and benzaldehyde decarbonylase.
[0085] In an additional embodiment, the invention provides a non-naturally occurring microbial
organism having a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate,
p-toluate, terephthalate, toluene, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, or
benzene pathway, wherein the non-naturally occurring microbial organism comprises
at least one exogenous nucleic acid encoding an enzyme or protein that converts a
substrate to a product. For example, in a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate
pathway, the substrates and products can be selected from the group consisting of
glyceraldehyde-3-phosphate and pyruvate to 1-deoxy-D-xylulose-5-phosphate; 1-deoxy-D-xylulose-5-phosphate
to C-methyl-D-erythritol-4-phosphate; and C-methyl-D-erythritol-4-phosphate to (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate
(see Example III and Figure 5). In another embodiment, a
p-toluate pathway can comprise substrates and products selected from (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate
to 2,4-dihydroxy-5-methyl-6-[(phosphonooxy)methyl]oxane-2-carboxylate; 2,4-dihydroxy-5-methyl-6-[(phosphonooxy)methyl]oxane-2-carboxylate
to 1,3-dihydroxy-4-methyl-5-oxocyclohexane-1-carboxylate; 1,3-dihydroxy-4-methyl-5-oxocyclohexane-1-carboxylate
to 5-hydroxy-4-methyl-3-oxocyclohex-1-ene-1-carboxylic acid; 5-hydroxy-4-methyl-3-oxocyclohex-1-ene-1-carboxylic
acid to 3,5-dihydroxy-4-methylcyclohex-1-ene-1-carboxylate; 3,5-dihydroxy-4-methylcyclohex-1-ene-1-carboxylate
to 5-hydroxy-4-methyl-3-(phosphonooxy)cyclohex-1-ene-1-carboxylate; 5-hydroxy-4-methyl-3-(phosphonooxy)cyclohex-1-ene-1-carboxylate
to 5-[(1-carboxyeth-1-en-1-yl)oxy]-4-methyl-3-(phosphonooxy)cyclohex-1-ene-1-carboxylate;
5-[(1-carboxyeth-1-en-1-yl)oxy]-4-methyl-3-(phosphonooxy)cyclohex-1-ene-1-carboxylate
to 3-[(1-carboxyeth-1-en-1-yl)oxy]-4-methylcyclohexa-1,5-diene-1-carboxylate; and
3-[(1-carboxyeth-1-en-1-yl)oxy]-4-methylcyclohexa-1,5-diene-1-carboxylate to
p-toluate (see Example IV and Figure 6). In still another embodiment, a terephthalate
pathway can comprise substrates and products selected from
p-toluate to 4-carboxybenzyl alcohol; 4-carboxybenzyl alcohol to 4-carboxybenzaldehyde;
and 4-carboxybenzaldehyde to and terephthalic acid (see Example V and Figure 7). In
another embodiment, a toluene pathway can comprise substrates and products selected
fromp
p-toluate to toluene;
p-toluate to
p-methyl benzoyl-CoA;
p-methyl benzoyl-CoA to
p-methylbenzoyloxy phosphate or
p-methylbenzadehyde;
p-methylbenzoyloxy phosphonate to
p-methylbenzaldehyde; and
p-methylbenzaldehyde to toluene (see Example VII and Figure 11). In another embodiment,
a 2H4OP pathway can comprise substrates and products selected from erythrose-4-phosphate
to (2,4-dioxobutoxy)phosphonate; and (2,4-dioxobutoxy)phosphonate to 2H4OP (see Example
VI and Figure 8). In another embodiment, a benzoate pathway can comprise substrates
and products selected from (2-hydroxy-4-oxobutoxy)phosphonate to 2,4-dihydroxy-6-[(phosphonooxy)methyl]oxane-2-carboxylate;
2,4-dihydroxy-6-[(phosphonooxy)methyl]oxane-2-carboxylate to 1,3-dihydroxy-5-oxocyclohexane-1-carboxylate;
1,3-dihydroxy-5-oxocyclohexane-1-carboxylate to 5-hydroxy-3-oxocyclohex-1-ene-1-carboxytate;
5-hydroxy-3-oxocyclohex-1-ene-1-carboxylate to 3,5-dihydroxycyclohex-1-ene-1-carboxylate,
3,5-dihydroxycyclohex-1-ene-1-carboxylate to 5-hydroxy-3-(phosphonooxy)cyclohex-1-ene-1-carboxylate;
5-hydroxy-3-(phosphonooxy)cyclohex-1-ene-1-carboxylate to 5-[(1-carboxyeth-1-en-1-yl)oxy]-3-(phosphonooxy)cyclohex-1-ene-1-carboxylate;
5-[(1-carboxyeth-1-en-1-yl)oxy]-3-(phosphonooxy)cyclohex-1-ene-1-carboxylate to 3-[(1-carboxyeth-1-en-1-yl)oxy]cyclohexa-1,5-diene-1-carboxylate;
and 3-[(1-carboxyeth-1-en-1-yl)oxy]cyclohexa-1,5-diene-1-carboxylate to benzoate (see
Example VI and Figure 9). In another embodiment, a benzene pathway can comprise substrates
and products selected from benzoate to benzene; benzoate to benzoyl-CoA, (benzoyloxy)phosphonate
or benzaldehyde; benzoyl-CoA to (benzoyloxy)phosphonate or benzaldehyde; (benzoyloxy)phosphonate
to benzaldehyde; and benzaldehyde to benzene. One skilled in the art will understand
that these are merely exemplary and that any of the substrate-product pairs disclosed
herein suitable to produce a desired product and for which an appropriate activity
is available for the conversion of the substrate to the product can be readily determined
by one skilled in the art based on the teachings herein.
[0086] As disclosed herein, a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway is exemplified
in Figure 5 (see Example III). Therefore, in addition to a microbial organism containing
a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway that produces (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate,
the invention additionally provides a non-naturally occurring microbial organism comprising
at least one exogenous nucleic acid encoding a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate
pathway enzyme, where the microbial organism produces a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate
pathway intermediate, for example, 1-deoxy-D-xylulose-5-phosphate or C-methyl-D-erythritol-4-phosphate.
Similarly, the invention also provides a non-naturally occurring microbial organism
containing a
p-toluate pathway that produces
p-toluate, wherein the non-naturally occurring microbial organism comprises at least
one exogenous nucleic acid encoding a
p-toluate pathway enzyme, where the microbial organism produces a
p-toluate pathway intermediate, for example, 2,4-dihydroxy-5-methyl-6-[(phosphonooxy)methyl]oxane-2-carboxylate,
1,3-dihydroxy-4-methyl-5-oxocyclohexane-1-carboxylate, 5-hydroxy-4-methyl-3-oxocyclohex-1-ene-1-carboxylate,
3,5-dihydroxy-4-methylcyclohex-1-ene-1-carboxylate, 5-hydroxy-4-methyl-3-(phosphonooxy)cyclohex-1-ene-1-carboxylate,
5-[(1-carboxyeth-1-en-1-yl)oxy]-4-methyl-3-(phosphonooxy)cyclohex-1-ene-1-carboxylate,
or 3-[(1-carboxyeth-1-en-1-yl)oxy]-4-methylcyclohexa-1,5-diene-1-carboxylate. Further,
the invention additionally provides a non-naturally occurring microbial organism containing
a terephthalate pathway enzyme, where the microbial organism produces a terephthalate
pathway intermediate, for example, 4-carboxybenzyl alcohol or 4-carboxybenzaldehyde.
[0087] Similarly, the invention also provides a non-naturally occurring microbial organism
containing a toluene pathway that produces toluene, wherein the non-naturally occurring
microbial organism comprises at least one exogenous nucleic acid encoding a toluene
pathway enzyme, where the microbial organism produces a toluene pathway intermediate,
for example,
p-methylbenzoyl-CoA, (
p-methylbenzoyloxy)phosphonate, or
p-methylbenzaldehyde.
[0088] Similarly, the invention also provides a non-naturally occurring microbial organism
containing a benzene pathway that produces benzene, wherein the non-naturally occurring
microbial organism comprises at least one exogenous nucleic acid encoding a benzene,
pathway enzyme, where the microbial organism produces a benzene pathway intermediate,
for example, benzoyl-CoA, (benzoyloxy)phosphonate, and benzaldehyde (Figure 10).
[0089] Similarly, the invention also provides a non-naturally occurring microbial organism
containing a benzoate pathway that produces benzoate, wherein the non-naturally occurring
microbial organism comprises at least one exogenous nucleic acid encoding a benzoate
pathway enzyme, where the microbial organism produces a benzoate pathway intermediate,
for example, (2-hydroxy-4-oxobutoxy)phosphonate, 2,4-dihydroxy-6-[(phosphonooxy)methyl]oxane-2-carboxylate,
1,3-dihydroxy-5-oxocyclohexane-1-carboxylate, 5-hydroxy-3-oxocyclohex-1-ene-1-carboxylate,
3,5-dihydroxycyclohex-1-ene-1-carboxylate, 5-hydroxy-3-(phosphonooxy)cyclohex-1-ene-1-carboxylate,
5-[(1-carboxyeth-1-en-1-yl)oxy]-3-(phosphonooxy)cyclohex-1-ene-1-carboxylate, and
3-[(1-carboxyeth-1-en-1-yl)oxy]cyclohexa-1,5-diene-1-carboxylate (Figure 9).
[0090] Thus, the invention provides a non-naturally occurring microbial organism containing
at least one exogenous nucleic acid encoding an enzyme or protein, where the enzyme
or protein converts the substrates and products of a toluene, benzene, p-toluate,
terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)psosphonate, (2-hydroxy-4-oxobutoxy)phosphonate,
benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene pathway, such
as those shown in Figures 1-11.
[0091] While generally described herein as a microbial organism that contains a toluene,
benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate,
benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene pathway, it is
understood that the invention additionally provides a non-naturally occurring microbial
organism comprising at least one exogenous nucleic acid encoding a toluene, benzene,
p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate,
benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene pathway enzyme
expressed in a sufficient amount to produce an intermediate of a toluene, benzene,
p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate,
benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol or 1,3-butadiene pathway. For example,
as disclosed herein, toluene, benzene, styrene, and 1,3-butadiene pathways are exemplified
in Figures 1-23. Therefore, in addition to a microbial organism containing a toluene,
benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate,
benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene pathway that produces
toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate,
(2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol
or 1,3-butadiene, the invention additionally provides a non-naturally occurring microbial
organism comprising at least one exogenous nucleic acid encoding a toluene, benzene,
p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate,
benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene pathway enzyme,
where the microbial organism produces a toluene, benzene, p-toluate, terephthalate,
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate,
styrene, 2,4-pentadienoate, 3-butene-1ol or 1,3-butadiene pathway intermediate, for
example, phenylpyruvate, phenylacetaldehyde, phenylacetate, 3-oxo-3-phenylpropionyl-CoA,
[(3-oxo-3-phenylpropionyl)oxy] phosphonate, benzoyl-acetate, acetophenone, 1-phenylethanol,
DXP, 2ME4P, benzoyl-CoA, (benzoyloxy)phosphonate, benzaldehyde,
trans-2,4-pentadienoate, and
cis-2,4-pentadienoate, as well as any shikimate pathway intermediate shown in Figures
6 and 9.
[0092] It is understood that any of the pathways disclosed herein, as described in the Examples
and exemplified in the Figures, including the pathways of Figures 1-11, can be utilized
to generate a non-naturally occurring microbial organism that produces any pathway
intermediate or product, as desired. As disclosed herein, such a microbial organism
that produces an intermediate can be used in combination with another microbial organism
expressing downstream pathway enzymes to produce a desired product. However, it is
understood that a non-naturally occurring microbial organism that produces a toluene,
benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate,
benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene pathway intermediate
can be utilized, to produce the intermediate as a desired product.
[0093] The invention is described herein with general reference to the metabolic reaction,
reactant or product thereof, or with specific reference to one or more nucleic acids
or genes encoding an enzyme associated with or catalyzing, or a protein associated
with, the referenced metabolic reaction, reactant or product. Unless otherwise expressly
stated herein, those skilled in the art will understand that reference to a reaction
also constitutes reference to the reactants and products of the reaction. Similarly,
unless otherwise expressly stated herein, reference to a reactant or product also
references the reaction, and reference to any of these metabolic constituent also
references the gene or genes encoding the enzymes that catalyze or proteins involved
in the referenced reaction, reactant or product. Likewise, given the well known fields
of metabolic biochemistry, enzymology and genomics, reference herein to a gene or
encoding nucleic acid also constitutes a reference to the corresponding encoded enzyme
and the reaction it catalyzes or a protein associated with the reaction as well as
the reactants and products of the reaction.
[0094] The non-naturally occurring microbial organisms of the invention can be produced
by introducing expressible nucleic acids encoding one or more of the enzymes or proteins
participating in one or more toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate,
(2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol
or 1,3-butadiene biosynthetic pathways. Depending on the host microbial organism chosen
for biosynthesis, nucleic acids for some or all of a particular toluene, benzene,
p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate,
benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol or 1,3-butadiene biosynthetic pathway
can be expressed. For example, if a chosen host is deficient in one or more enzymes
or proteins for a desired biosynthetic pathway, then expressible nucleic acids for
the deficient enzyme(s) or protein(s) are introduced into the host for subsequent
exogenous expression. Alternatively, if the chosen host exhibits endogenous expression
of some pathway genes, but is deficient in others, then an encoding nucleic acid is
needed for the deficient enzyme(s) or protein(s) to achieve toluene, benzene, p-toluate,
terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate,
benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol or 1,3-butadiene biosynthesis.
Thus, a non-naturally occurring microbial organism of the invention can be produced
by introducing exogenous enzyme or protein activities to obtain, a desired biosynthetic
pathway or a desired biosynthetic pathway can be obtained by introducing one or more
exogenous enzyme or protein activities that, together with one or more endogenous
enzymes or proteins, produces a desired product such as toluene, benzene, p-toluate,
terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate,
benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol or 1,3-butadiene
[0095] Host microbial organisms can be selected from, and the non-naturally occurring microbial
organisms generated in, for example, bacteria, yeast, fungus or any of a variety of
other microorganisms applicable to fermentation processes. Exemplary bacteria include
species selected from
Escherichia coli, Klebsiella oxytoca, Anaerobiospirillum succiniciproducens, Actinobacillus
succinogenes, Mannheimia succiniciproducens, Rhizobium etli,
Bacillus subtilis, Corynebacterium glutamicum, Gluconobacter oxydans, Zymomonas mobilis,
Lactococcus lactis, Lactobacillus plantarum, Streptomyces coelicolor, Clostridium
acetobutylicum, Pseudomonas fluorescens, and
Pseudomonas putida. Exemplary yeasts or fungi include species selected from
Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces
marxianus, Aspergillus terreus,
Aspergillus niger, Pichia pastoris, Rhizopus arrhizus, Rhizobus oryzae, and the like.
E. coli is a particularly useful host organisms since it is a well characterized microbial
organism suitable for genetic engineering. Other particularly useful host organisms
include yeast such as
Saccharomyces cerevisiae. It is understood that any suitable microbial host organism can be used to introduce
metabolic and/or genetic modifications to produce a desired product.
[0096] Depending on the toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate,
(2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol
or 1,3-butadiene biosynthetic pathway constituents of a selected host microbial organism,
the non-naturally occurring microbial organisms of the invention will include at least
one exogenously expressed toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate,
(2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol
or 1,3-butadiene pathway-encoding nucleic acid and up to all encoding nucleic acids
for one or more toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate,
(2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol
or 1,3-butadiene biosynthetic pathways. For example, toluene, benzene, p-toluate,
terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate,
benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol or 1,3-butadiene biosynthesis can
be established in a host deficient in a pathway enzyme or protein through exogenous
expression of the corresponding encoding nucleic acid. In a host deficient in all
enzymes or proteins of a toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate,
(2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol
or 1,3-butadiene pathway, exogenous expression of all enzyme or proteins in the pathway
can be included, Although it is understood that all enzymes or proteins of a pathway
can be expressed even if the host contains at least one of the pathway enzymes or
proteins. For example, exogenous expression of all enzymes or proteins in a pathway
for production of toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate,
(2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol
or 1,3-butadiene can be included, such as phenylalanine aminotransferase and/or phenylalanine
oxidoreductase (deaminating), phenylpyruvate decarboxylase, phenylacetaldehyde dehydrogenase
and/or oxidase, phenylpyruvate oxidase, phenylacetate decarboxylase, phenylacetaldehyde
decarbonylase, phenylalanine benzene-lyase, benzoyl-CoA acetyltransferase, 3-oxo-3-phenylpropionyl-CoA
synthetase, transferase and/or hydrolase, benzoyl-acetate decarboxylase, acetophenone
reductase, 1-phenylethanol dehydratase, phosphotrans-3-oxo-3-phenylpropionylase, benzoyl-acetate
kinase,
trans, trans-muconate decarboxylase,
cis,
trans-muconate
cis-decarboxylase,
cis,
trans-muconate
trans-decarboxylase,
cis,
cis-muconate decarboxylase,
trans-2,4-pentadienoate decarboxylase, and
cis-2,4-pentadienoate decarboxylase.
[0097] For example, all enzymes in a
p-toluate pathway can be included, such as 2-dehydro-3-deoxyphosphoheptonate synthase;
3-dehydroquinate synthase; 3-dehydroquinate dehydratase; shikimate dehydrogenase;
shikimate kinase; 3-phosphoshikimate-2-carboxyvinyltransferase; chorismate synthase;
and chorismate lyase. In addition, all enzymes in a terephthalate pathway can be included,
such as
p-toluate methyl-monooxygenase reductase; 4-carboxybenzyl alcohol dehydrogenase; and
4-carboxybenzyl aldehyde dehydrogenase. Furthermore, all enzymes in a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate
pathway can be included, such as 2-C-methyl-D-erythritol-4-phosphate dehydratase,
1-deoxyxylulose-5-phosphate synthase and 1-deoxy-D-xylulose-5-phosphate reductoisomerase.Likewise,
all enzymes in a toluene pathway can be included, such as
p-methylbenzoyl-CoA synthetase, transferase and/or hydrolase,
p-toluate reductase
, p-methylbenzaldehyde decarbonylase,
p-methylbenzoyl-CoA reductase,
p-toluate decarboxylase, phosphotrans-
p-methylbenzoylase, (
p-methylbenzoyloxy)phosphonate reductase (dephosphorylating), and
p-toluate kinase.
[0098] Likewise, all enzymes in a (2-hydroxy-4-oxobutoxy)phosphonate pathway can be included,
such as erythrose-4-phosphate dehydratase and (2,4-dioxobutoxy)phosphonate reductase.
[0099] Likewise, all enzymes in a benzozate pathway can be included, such as 2-dehydro-3-deoxyphosphoheptonate
synthase, 3-dehydroquinate synthase, 3-dehydroquinate dehydratase, shikimate dehydrogenase,
shikimate kinase, 3-phosphoshikimate-2-carboxyvinyltransferase, chorismate synthase,
and chorismate lyase.
[0100] Likewise, all enzymes in a benzene pathway can be included, such as benzoyl-CoA synthetase,
transferase and/or hydrolase, benzoate reductase, benzaldehyde decarbonylase, benzoyl-CoA
reductase, benzoate decarboxylase, phosphotransbenzoylase, (benzoyloxy)phosphonate
reductase (dephosphorylating), and benzoate kinase.
[0101] Given the teachings and guidance provided herein, those skilled in the art will understand
that the number of encoding nucleic acids to introduce in an expressible form will,
at least, parallel the toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate,
(2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol
or 1,3-butadiene pathway deficiencies of the selected host microbial organism. Therefore,
a non-naturally occurring microbial organism of the invention can have one, two, three,
four, five, six, seven, eight, that is, up to all nucleic acids encoding the enzymes
or proteins constituting a toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate,
(2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol
or 1,3-butadiene biosynthetic pathway disclosed herein. In some embodiments, the non-naturally
occurring microbial organisms also can include other genetic modifications that facilitate
or optimize toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate,
(2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol
or 1,3-butadiene biosynthesis or that confer other useful functions onto the host
microbial organism. One such other functionality can include, for example, augmentation
of the synthesis of one or more of the toluene, benzene, p-toluate, terephthalate,
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate,
styrene, 2,4-pentadienoate, 3-butene-1ol or 1,3-butadiene pathway precursors such
as phenylalanine, phenylpyruvate, phenylacetaldehyde, phenylacetate, benzoyl-CoA,
3-oxo-3-phenylpropionyl-CoA, [(3-oxo-3-phenylpropionyl)oxy]phosphonate, benzoyl acetate,
acetophenone, 1-phenylethanol,
trans,trans-muconate,
cis,trans-muconate,
cis,cis-muconate,
trans-2,4-pentadienoate,
cis-2,4-pentadienoate, 4-hydroxy-2-oxovalerate, 2-oxopentenoate, 2-hydroxypentenoate,
2,4-dihydroxypentanoate, 4-hydroxypent-2-enoate, 2,4-diaminopentanoate, AKP, acetylacrulate,
2,4-dioxopentanoate, 2-hydroxy-4-oxo-pentanoate, 2-amino-4-hydroxypentanoate, 3,5-diaminopentanoate,
5-aminopent-2-enoate, 3-amino-5-oxopentanoate, 5-oxopent-2-enoate, 5-hydroxypent-2-enoate,
3-amino-5-hydroxypentanoate, 3-HP-CoA, acryloyl-CoA, 3-oxo-5-hydroxypentanoyl-CoA,
3-oxo-5-hydroxypentanoate, 3,5-dihydroxypentanoate, 3,5-dihydroxypentanoyl-CoA, 5-hydroxypent-2-enoyl-CoA,
2,4-pentadienoyl-CoA, 3-oxopent-4-enoyl-CoA, 3-hydroxypent-4-enoyl-CoA, 3-oxopent-4-enoate,
and 3-hydroxypent-4-enoate glyceraldehyde-3-phosphate, pyruvate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate
or p-toluate. Furthermore, as disclosed herein, multiple pathways can be included
in a single organism such as the pathway to produce p-toluate (Figure 6), terephthalate
(Figure 7) toluene (Figure 11) and (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate (Figure
5), as desired, or 2H4OP (Figure 8), benzoate (Figure 9) and benzene (Figure 10).
[0102] Generally, a host microbial organism is selected such that it produces the precursor
of a toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate,
(2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol
or 1,3-butadiene pathway, either as a naturally produced molecule or as an engineered
product that either provides
de novo production of a desired precursor or increased production of a precursor naturally
produced by the host microbial organism. For example,
cis,cis-muconate is produced naturally in a host organism such as
E. coli. As a further example, glyceraldehyde-3-phosphate and phosphoenolpyruvate are produced
naturally in a host organism such as E. coli. A host organism can be engineered to
increase production of a precursor, as disclosed herein. In addition, a microbial
organism that has been engineered to produce a desired precursor can be used as a
host organism and further engineered to express enzymes or proteins of a toluene,
benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate,
benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol or 1,3-butadiene pathway.
[0103] In some embodiments, a non-naturally occurring microbial organism of the invention
is generated from a host that contains the enzymatic capability to synthesize toluene,
benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate,
benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol or 1,3-butadiene. In this specific
embodiment it can be useful to increase the synthesis or accumulation of a toluene,
benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate,
benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol or 1,3-butadiene pathway product
to, for example, drive toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate,
(2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol
or 1,3-butadiene pathway reactions toward toluene, benzene, p-toluate, terephthalate
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate,
styrene, 2,4-pentadienoate, 3-butene-1ol or 1,3-butadiene production. Increased synthesis
or accumulation can be accomplished by, for example, overexpression of nucleic acids
encoding one or more of the above-described toluene, benzene, p-toluate, terephthalate,
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate,
styrene, 2,4-pentadienoate, 3-butene-1ol or 1,3-butadiene pathway enzymes or proteins.
Over expression the enzyme or enzymes and/or protein, or proteins of the toluene,
benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate,
benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol or 1,3-butadiene pathway can occur,
for example, through exogenous expression of the endogenous gene or genes, or through
exogenous expression of the heterologous gene or genes. Therefore, naturally occurring
organisms can be readily generated to be non-naturally occurring microbial organisms
of the invention, for example, producing toluene, benzene, p-toluate, terephthalate,
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate,
styrene, 2,4-pentadienoate, 3-butene-1ol or 1,3-butadiene , through overexpression
of one, two, three, four, five, six, seven, eight, that is, up to all nucleic acids
encoding toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate,
(2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol
or 1,3-butadiene biosynthetic pathway enzymes or proteins. In addition, a non-naturally
occurring organism can be generated by mutagenesis of an endogenous gene that results
in an increase in activity of an enzyme in the toluene, benzene, p-toluate, terephthalate,
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate,
styrene, 2,4-pentadienoate, 3-butene-1ol or 1,3-butadiene biosynthetic pathway.
[0104] In particularly useful embodiments, exogenous expression of the encoding nucleic
acids is employed. Exogenous expression confers the ability to custom tailor the expression
and/or regulatory elements to the host and application to achieve a desired expression
level that is controlled by the user. However, endogenous expression also can be utilized
in other embodiments such as by removing a negative regulatory effector or induction
of the gene's promoter when linked to an inducible promoter or other regulatory element.
Thus, an endogenous gene having a naturally occurring inducible promoter can be up-regulated
by providing the appropriate inducing agent, or the regulatory region of an endogenous
gene can be engineered to incorporate an inducible regulatory element, thereby allowing
the regulation of increased expression of an endogenous gene at a desired time. Similarly,
an inducible promoter can be included as a regulatory element for an exogenous gene
introduced into a non-naturally occurring microbial organism.
[0105] It is understood that, in methods of the invention, any of the one or more exogenous
nucleic acids can be introduced into a microbial organism to produce a non-naturally
occurring microbial organism of the invention. The nucleic acids can be introduced
so as to confer, for example, a toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methy)-4-oxobutoxy)phosphonate,
(2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol
or 1,3-butadiene biosynthetic pathway onto the microbial organism. Alternatively,
encoding nucleic acids can be introduced to produce an intermediate microbial organism
having the biosynthetic capability to catalyze some of the required reactions to confer
toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate,
(2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol
or 1,3-butadiene biosynthetic capability. For example, a non-naturally occurring microbial
organism having a toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate,
(2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol
or 1,3-butadiene biosynthetic pathway can comprise at least two exogenous nucleic
acids encoding desired enzymes or proteins, such as the combination of phenylalanine
aminotransferase and/or phenylalanine oxidoreductase (deaminating) and phenylpyruvate
decarboxylase, phenylalanine aminotransferase and/or phenylalanine oxidoreductase
(deaminating) and phenylacetaldehyde dehydrogenase and/or oxidase, phenylalanine aminotransferase
and/or phenylalanine oxidoreductase (deaminating) and phenylpyruvate oxidase, phenylpyruvate
oxidase and phenylacetate decarboxylase, phenylalanine aminotransferase and/or phenylalanine
oxidoreductase (deaminating) and phenylacetate decarboxylase, phenylalanine aminotransferase
and/or phenylalanine oxidoreductase (deaminating) and phenylacetaldehyde decarbonylase,
phenylpyruvate decarboxylase and phenylacetaldehyde dehydrogenase, phenypyruvate decarboxylase
and phenylacetate decarboxylase, phenylpyruvate decarboxylase and phenylacetaldehyde
decarbonylase, and phenylacetaldehyde dehydrogenase and/or oxidase and phenylacetate
decarboxylase, in a toluene pathway. Similarly, in a styrene pathway the combination
of at least two exogenous nucleic acids can include benzoyl-CoA acetyltransferase
and 3-oxo-3-phenylpropionyl-CoA synthetase, benzoyl-CoA acetyltransferase and benzoyl-acetate
decarboxylase, benzoyl-CoA acetyltransferase and acetophenone reductase, benzoyl-CoA
acetyltransferase and 1-phenylethanol dehydratase, benzoyl-CoA acetyltransferase and
phosphotrans-3-oxo-3-phenylpropionylase, benzoyl-CoA acetyltransferase and benzoyl-acetate
kinase, 3-oxo-3-phenylpropionyl-CoA synthetase and benzoyl-acetate decarboxylase,
3-oxo-3-phenylpropionyl-CoA synthetase and acetopheonone reductase, 3-oxo-3-phenylpropionyl-CoA
synthetase and 1-phenylethanol dehydratase, and so on.
[0106] Similarly, in a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway, a combination
of the enzymes expressed can be a combination of 2-C-methyl-D-erythritol-4-phosphate
dehydratase and 1-deoxyxylulose-5-phosphate synthase, or 2-C-methyl-D-erythritol-4-phosphate
dehydratase and 1-deoxy-D-xylutose-5-phosphate reductoisomerase. In a p-toluate pathway,
a combination of the enzymes expressed can be a combination of 2-dehydro-3-deoxyphosphoheptonate
synthase and 3-dehydroquinate dehydratase; shikimate kinase and 3-phosphoshikimate-2-carboxyvinyltransferase;
shikimate kinase and shikimate dehydrogenase and, and the like. Similarly, in a terephthalate
pathway, a combination of the expressed enzymes can be p-toluate methyl-monooxygenase
reductase and 4-carboxybenzyl alcohol dehydrogenase; or 4-carboxybenzyl alcohol dehydrogenase
and 4-carboxybenzyl aldehyde dehydrogenase, and the like. Thus, it is understood that
any combination of two or more enzymes or proteins of a biosynthetic pathway can be
included in a non-naturally occurring microbial organism of the invention.
[0107] Similarly, it is understood that any combination of three or more enzymes or proteins
of a biosynthetic pathway can be included in a non-naturally occurring microbial organism
of the invention, and so forth, as desired, so long as the combination of enzymes
and/or proteins of the desired biosynthetic pathway results in production of the corresponding
desired product. Such combination of three enzymes can include, for example, 3-dehydroquinate
synthase, shikimate dehydrogenase and shikimate kinase; shikimate kinase, chorismate
synthase and chorismate lyase; 3-dehydroquinate dehydratase, chorismate synthase and
chorismate lyase, and so forth, as desired, so long as the combination of enzymes
and/or proteins of the desired biosynthetic pathway results in production of the corresponding
desired product.
[0108] Similarly, any combination of four, five, six, or more enzymes or proteins of a biosynthetic
pathway as disclosed herein can be included in a non-naturally occurring microbial
organism of the invention, as desired, so long as the combination of enzymes and/or
proteins of the desired biosynthetic pathway results in production of the corresponding
desired product.
[0109] In addition to the biosynthesis of toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate,
(2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol
or 1,3-b-butadiene as described herein, the non-naturally occurring microbial organisms
and methods of the invention also can be utilized in various combinations with each
other and with other microbial organisms and methods well known in the art to achieve
product biosynthesis by other routes. For example, one alternative to produce toluene,
benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate,
benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol or 1,3-butadiene other than use
of the toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate,
(2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol
or 1,3-butadiene producers is through addition of another microbial organism capable
of converting a toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate,
(2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol
or 1,3-butadiene pathway intermediate to toluene, benzene, p-toluate, terephthalate,
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate,
styrene, 2,4-pentadienoate, 3-butene-1ol or 1,3-butadiene . One such procedure includes,
for example, the fermentation of a microbial organism that produces a toluene, benzene,
p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate,
benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol or 1,3-butadiene pathway intermediate.
The toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate,
(2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol
or 1,3-butadiene pathway intermediate can then be used as a substrate for a second
microbial organism that converts the toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate,
(2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol
or 1,3-butadiene pathway intermediate to toluene, benzene, p-toluate, terephthalate,
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate,
styrene, 2,4-pentadienoate, 3-butene-1ol or 1,3-butadiene . The toluene, benzene,
p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate,
benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol or 1,3-butadiene pathway intermediate
can be added directly to another culture of the second organism or the original culture
of the toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate,
(2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol
or 1,3-butadiene pathway intermediate producers can be depleted of these microbial
organisms by, for example, cell separation, and then subsequent addition of the second
organism to the fermentation broth can be utilized to produce the final product without
intermediate purification steps.
[0110] In other embodiments, the non-naturally occurring microbial organisms and methods
of the invention can be assembled in a wide variety of subpathways to achieve biosynthesis
of, for example, toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate,
(2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol
or 1,3-butadiene . In these embodiments, biosynthetic pathways for a desired product
of the invention can be segregated into different microbial organisms, and the different
microbial organisms can be co-cultured to produce the final product. In such a biosynthetic
scheme, the product of one microbial organism is the substrate for a second microbial
organism until the final product is synthesized. For example, the biosynthesis of
toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate,
(2-hydroxy-4-oxobutoxy)phosphonate, styrene, 2,4-pentadienoate, 3-butene-1ol or 1,3-butadiene
can be accomplished by constricting a microbial organism that contains biosynthetic
pathways for conversion of one pathway intermediate to another pathway intermediate
or the product. Alternatively, toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate,
(2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol
or 1,3-butadiene also can be biosynthetically produced from microbial organisms through
co-culture or co-fermentation using two organisms in the same vessel, where the first
microbial organism produces a toluene, benzene, p-toluate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate,
(2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol
or 1,3-butadiene intermediate and the second microbial organism converts the intermediate
to toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate,
(2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol
or 1,3-butadiene.
[0111] Given the teachings and guidance provided herein, those skilled in the art will understand
that a wide variety of combinations permutations exist for the non-naturally occurring
microbial organisms and methods of the invention together with other microbial organisms,
with the co-culture of other non-naturally occurring microbial organisms having subpathways
and with combinations of other chemical and/or biochemical, procedures well known
in the art to produce toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate,
(2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol
or 1,3-butadiene .
[0112] Sources of encoding nucleic acids for a toluene, benzene, p-toluate, terephthalate,
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate,
styrene, 2,4-pentadienoate, 3-butene-1ol or 1,3-butadiene pathway enzyme or protein
can include, for example, any species where the encoded gene product is capable of
catalyzing the referenced reaction. Such species include both prokaryotic and eukaryotic
organisms including, but not limited to, bacteria, including archaea and cubacteria,
and eukaryotes, including yeast, plant, insect, animal, and mammal, including human.
Exemplary species for such sources include, for example,
Escherichia coli, Mycobacterium tuberculosis, Agrobacterium tumefaciens, Becillus
subtilis, Synechocystis species,
Arabidopsis thaliana, Zymomonas mobilis, Klebsiella oxytoca, Salmonella typhimurium,
Salmonella typhi, Lactobacullus collinoides, Klebsiella pneumoniae, Clostridium pasteuranum,
Citrobacter freundii, Clostridium butyricum, Roseburia inulinivorans, Sulfolobus solfataricus,
Neurospora crassa, Sinorhizobium fredii, Helicobacter pylori, Pyrococcus furiosus,
Haemophilus influenzae, Erwinia chrysanthemi, Staphylococcus aureus, Dunaliellea salina,
Streptococcus pneumoniae, Saccharomyces cerevisiae, Aspergillus nidulans, Pneumocystis
carinii, Streptomyces coelicolor, species from the genera
Burkholderia, Alcaligenes, Pseudomonas, Shingomonas and
Comamonas, for example,
Comamonas testosteroni, as well as other exemplary species disclosed herein or available as source organisms
for corresponding genes. However, with the complete genome sequence available for
now more than 550 species (with more than half of these available on public databases
such as the NCBI), including 395 microorganism genomes and a variety of yeast, fungi,
plant, and mammalian genomes, the identification of genes encoding the requisite toluene,
benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate,
benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol or 1,3-butadiene biosynthetic activity
for one or more genes in related or distant species, including for example, homologues,
orthologs, paralogs and nonorthologous gene displacements of known genes, and the
interchange of genetic alterations between organism is routine and well known in the
art. Accordingly, the metabolic alterations allowing biosynthesis of toluene, benzene,
p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate,
benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol or 1,3-butadiene described herein
with reference to a particular organism such as
E. coli can be readily applied to other microorganisms, including prokaryotic and eukaryotic
organisms alike. Given the teachings and guidance provided herein, those skilled in
the art will know that a metabolic alteration exemplified in one organism can be applied
equally to other organisms.
[0113] In some instances, such as when an alternative toluene, benzene, p-toluate, terephthalate,
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate,
styrene, 2,4-pentadienoate, 3-butene-1ol or 1,3-butadiene biosynthetic pathway exists
in an unrelated species, toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phospbonate,
(2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol
or 1,3-butadiene biosynthesis can be conferred onto the host species by, for example,
exogenous expression of a paralog or paralogs from the unrelated species that catalyzes
a similar, yet non-identical metabolic reaction to replace the referenced reaction.
Because certain differences among metabolic networks exist between different organisms,
those skilled in the art will understand that the actual gene usage between different
organisms may differ. However, given the teachings and guidance provided herein, those
skilled in the art also will understand that the teachings and methods of the invention
can be applied to all microbial organisms using the cognate metabolic alterations
to those exemplified herein to construct a microbial organism in a species of interest
that will synthesize toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate,
(2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol
or 1,3-butadiene.
[0114] Methods for constructing and testing the expression levels of a non-naturally occurring
toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate,
(2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol
or 1,3-butadiene -producing can be performed, for example, by recombinant and detection
methods well known in the art. Such methods can be found described in, for example,
Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor
Laboratory, New York (2001); and
Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore,
MD (1999).
[0115] Exogenous nucleic acid sequences involved in a pathway for production of toluene,
benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate,
benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol or 1,3-butadiene can be introduced
stably or transiently into a host cell using techniques well known in the art including,
but not limited to, conjugation, electroporation, chemical transformation, transduction,
transfection, and ultrasound transformation. For exogenous expression in
E. coli or other prokaryotic cells, some nucleic acid sequences in the genes or cDNAs of
eukaryotic nucleic acids can encode targeting signals such as an N-terminal mitochondrial
or other targeting signal, which can be removed before transformation into prokaryotic
host cells, if desired. For example, removal of a mitochondrial leader sequence led
to increased expression in
E. coli (
Hoffmeister et al., J. Biol. Chem. 280:4329-4338 (2005)). For exogenous expression in yeast or other eukaryotic cells, genes can be expressed
in the cytosol without the addition of leader sequence, or can be targeted to mitochondrion
or other organelles, or targeted for secretion, by the addition of a suitable targeting
sequence such as a mitochondrial targeting or secretion signal suitable for the host
cells. Thus, it is understood that appropriate modifications to a nucleic acid sequence
to remove or include a targeting sequence can be incorporated into an exogenous nucleic
acid sequence to impart desirable properties. Furthermore, genes can be subjected
to codon optimization with techniques well known in the art to achieve optimized expression
of the proteins.
[0116] An expression vector or vectors can be constructed to include one or more toluene,
benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate,
benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol or 1,3-butadiene biosynthetic pathway
encoding nucleic acids as exemplified herein operably linked to expression control
sequences functional in the host organism. Expression vectors applicable for use in
the microbial host organisms of the invention include, for example, plasmids, phage
vectors, viral vectors, episomes and artificial chromosomes, including vectors and
selection sequences or markers operable for stable integration into a host chromosome.
Additionally, the expression vectors can include one or more selectable marker genes
and appropriate expression control sequences. Selectable marker genes also can be
included that, for example, provide resistance to antibiotics or toxins, complement
auxotrophic deficiencies, or supply critical nutrients not in the culture media. Expression
control sequences can include constitutive and inducible promoters, transcription
enhancers, transcription terminators, and the like which are well known in the art.
When two or more exogenous encoding nucleic acids are to be co-expressed, both nucleic
acids can be inserted, for example, into a single expression vector or in separate
expression vectors. For single vector expression, the encoding nucleic acids can be
operationally linked to one common expression control sequence or linked to different
expression control sequences, such as one inducible promoter and one constitutive
promoter. The transformation of exogenous nucleic acid sequences involved in a metabolic
or synthetic pathway can be confirmed using methods well known in the art. Such methods
include, for example, nucleic acid analysis such as Northern blots or polymerase chain
reaction (PCR) amplification of mRNA, or immunoblotting for expression of gene products,
or other suitable analytical methods to test the expression of an introduced nucleic
acid sequence or its corresponding gene product. It is understood by those skilled
in the art that the exogenous nucleic acid is expressed is a sufficient amount to
produce the desired product, and it is further understood that expression levels can
be optimized to obtain sufficient expression using methods well known in the art and
as disclosed herein.
[0117] In some embodiments, the present invention provides a method for producing toluene
that includes culturing a non-naturally occurring microbial organism having a toluene
pathway. The toluene pathway includes at least one exogenous nucleic acid encoding
a toluene pathway enzyme expressed in a sufficient amount to produce toluene, under
conditions and for a sufficient period, of time to produce toluene. The toluene pathway
can be selected from (A) 1) one or both of phenylalanine aminotransferase and phenylalanine
oxidoreductase (deaminating), 2) phenylpyruvate decarboxylase, and 3) phenylacetaldehyde
decarbonylase; (B) 1) one or more of phenylalanine aminotransferase and phenylalanine
oxidoreductase (deaminating), 2) phenylpyruvate decarboxylase, 3) one or more of phenylacetaldehyde
dehydrogenase and phenylacetaldehyde oxidase, and 4) phenylacetate decarboxylase;
and (C) 1) one or more of phenylalanine aminotransferase and phenylalanine oxidoreductase
(deaminating), 2) phenylpyruvate oxidase, and 3) phenylacetate decarboxylase, as indicated
in the alternate pathways shown in Figure 1. In some embodiments, the method includes
culturing the non-naturally occurring microbial organism is in a substantially anaerobic
culture medium.
[0118] In some embodiments, methods of the invention for producing toluene include culturing
a non-naturally microbial organism, that includes two exogenous nucleic acids each
encoding a toluene pathway enzyme, three exogenous nucleic acids each encoding a toluene
pathway enzyme, four exogenous nucleic acids each encoding a toluene pathway enzyme,
five exogenous nucleic acids each encoding a toluene pathway, and so on. Exemplary
organisms having four exogenous nucleic acids can encode 1) phenylalanine aminotransferase,
2) phenylalanine oxidoreductase (deaminating), 3) phenylpyruvate decarboxylase, and
4) phenylacetaldehyde decarbonylase. Exemplary organisms having five exogenous nucleic
acids can encode 1) phenylalanine aminotransferase, 2) phenylalanine oxidoreductase
(deaminating), 3) phenylpyruvate decarboxylase, 4) phenylacetaldehyde dehydrogenase
and/or oxidase, and 5) phenylacetate decarboxylase. In some embodiments, methods of
the invention for producing toluene can include culturing a non-naturally occurring
microbial organism that has at least one exogenous nucleic acid that is a heterologous
nucleic acid.
[0119] In some embodiments, the present invention provides a method for producing benzene
that includes culturing a non-naturally occurring microbial organism having a benzene
pathway. The benzene pathway can include at least one exogenous nucleic acid encoding
a benzene pathway enzyme expressed in a sufficient amount to produce benzene, under
conditions and for a sufficient period of time to produce benzene. The benzene pathway
can include a phenylalanine benzene-lyase, as shown in Figure 2. In some embodiments,
the at least one exogenous nucleic acid is the phenylalanine benzene-lyase itself,
while in alternate embodiments, the at least one exogenous nucleic acid can affect
the production of the precursor metabolite phenylalanine. In some embodiments the
at least one exogenous nucleic acid of the benzene pathway is a heterologous nucleic
acid. In some embodiments, methods of the invention for producing benzene can include
culturing a non-naturally occurring microbial organism that is in a substantially
anaerobic culture medium.
[0120] In some embodiments, the present invention provides a method for producing styrene
that includes culturing a non-naturally occurring microbial organism having a styrene
pathway. The styrene pathway can include at least one exogenous nucleic acid encoding
a styrene pathway enzyme expressed in a sufficient amount to produce styrene, under
conditions and for a sufficient period of time to produce styrene. The styrene pathway
can be selected from (A) 1) benzoyl-CoA acetyltransferase, 2) one or more of 3-oxo-3-phenylpropionyl-CoA
synthetase, transferase, and hydrolase, 3) benzoyl-acetate decarboxylase, 4) acetopheone
reductase, and 5) 1-phenylethanol dehydratase; and (B) 1) benzoyl-CoA acetyltransferase,
2) phosphotrans-3-oxo-3-phenylpropionylase, 3) benzoyl-acetate kinase, 4) benzoyl-acetate
decarboxylase, 5) acetopheone reductase, and 6) 1-phenylethanol dehydratase, as indicated
in the alternate pathways in Figure 3. In some embodiments, methods of the invention
for producing styrene can include culturing a non-naturally occurring microbial organism
that is in a substantially anaerobic culture medium.
[0121] In some embodiments, methods of the invention for producing styrene include culturing
a non-naturally microbial organism, that includes two exogenous nucleic acids each
encoding a styrene pathway enzyme, three exogenous nucleic acids each encoding a styrene
pathway enzyme, four exogenous nucleic acids each encoding a styrene pathway enzyme,
five exogenous nucleic acids each encoding a styrene pathway enzyme, six erogenous
nucleic acids each encoding a styrene pathway enzyme, and so on. An exemplary organism
having five exogenous nucleic acids can encode 1) benzoyl-CoA acetyltransferase, 2)
one of 3-oxo-3-phenylpropionyl-CoA synthetase, transferase, and hydrolase, 3) benzoyl-acetate
decarboxylase, 4) acetopheotic reductase, and 5) 1-phenylethanol dehydratase. An exemplary
organism having six six exogenous nucleic acids encode 1) benzoyl-CoA acetyltransferase,
2) phosphotrans-3-oxo-3-phenylpropionylase, 3) benzoyl-acetate kinase, 4) benzoyl-acetate
decarboxylase, 5) acetopheone reductase, and 6) 1-phenylethanol dehydratase. In some
embodiments, methods of the present, invention for producing styrene can include culturing
a non-naturally occurring microbial organism in which at least one exogenous nucleic
acid is a heterologous nucleic acid.
[0122] In some embodiments, the present invention provides a method for producing 1,3-butadiene
that includes culturing a non-naturally occurring microbial organism having a 1,3-butadiene
pathway. The 1,3-butadiene pathway includes at least one exogenous nucleic acid encoding
a 1,3-butadiene pathway enzyme expressed in a sufficient amount to produce 1,3-butadiene,
under conditions and for a sufficient period of time to produce 1,3-butadiene. The
1,3-butadiene pathway can be selected from (A) 1)
trans, trans-muconate decarboxylase and 2)
trans-2,4-pentadienoate decarboxylase; (B) 1)
cis,
trans-muconate
cis-decarboxylase and 2)
trans-2,4-pentadienoate decarboxylase; (C) 1)
cis, trans-muconate
trans-decarboxylase 2)
cis-2,4-pentadienoate decarboxylase; and (D) 1)
cis,
cis-muconate decarboxylase and 2)
cis-2,4-pentadienoate decarboxylase, as indicated by the alternate pathways in Figure
4. In some embodiments, the method of producing 1,3-butadiene can include culturing
a non-naturally occurring microbial organism that is in a substantially anaerobic
culture medium.
[0123] In some embodiments, methods of the invention can include culturing a non-naturally
occurring microbial organism that has two exogenous nucleic acids each encoding a
1,3-butadiene pathway enzyme. Exemplary organisms having two exogenous nucleic acids
can include genes encoding a set of enzymes selected from (A) 1)
trans, trans-muconate decarboxylase and 2)
trans-2,4-pentadienoate decarboxylase; (B) 1)
cis, trans-muconate
cis-decarboxylase and 2)
trans-2,4-pentadienoate decarboxylase; (C) 1)
cis, trans-muconate
trans-decarboxylase 2)
cis-2,4-pentadienoate decarboxylase; and (D) 1)
cis, cis-muconate decarboxylase and 2)
cis-2,4-pentadienoate decarboxylase. In some embodiments, methods of the invention can
include culturing a non-naturally occurring microbial organism that has at least one
exogenous nucleic acid that is a heterologous nucleic acid.
[0124] The invention additionally provides a method for producing (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate,
comprising culturing the non-naturally occurring microbial organism containing a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate
pathway under conditions and for a sufficient period of time to produce (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate.
Such a microbial organism can have a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway
comprising at least one exogenous nucleic acid encoding a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate
pathway enzyme expressed in a sufficient amount, to produce (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate,
the (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway comprising 2-C-methyl-D-erythritol-4-phosphate
dehydratase (see Example III and Figure 5, step C). A (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate
pathway can optionally further comprise 1-deoxyxylulose-5-phosphate synthase and/or
1-deoxy-D-xylulose-5-phosphate reductoisomerase (see Example III and Figure 5, steps
A and B).
[0125] In another embodiment, the invention provides a method for producing
p-toluate, comprising culturing the non-naturally occurring microbial organism comprising
a
p-toluate pathway under conditions and for a sufficient period of time to produce
p-toluate. A
p-toluate pathway can comprise at least one exogenous nucleic acid encoding a
p-toluate pathway enzyme expressed in a sufficient amount to produce
p-toluate, the
p-toluate pathway comprising 2-dehydro-3-deoxyphosphoheptonate synthase; 3-dehydroquinate
synthase; 3-dehydroquinate dehydratase; shikimate dehydrogenase; shikimate kinase;
3-phosphoshikimate-2-carboxyvinyltransferase; chorismate synthase; and/or chorismate
lyase (see Example IV and Figure 6, steps A-H). In another embodiment, a method of
the invention can utilize a non-naturally occurring microbial organism that further
comprises a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway (see Example III and
Figure 5). Such a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway can comprise
2-C-methyl-D-erythritol-4-phosphate dehydratase, 1-deoxyxylulose-5-phosphate synthase
and/or 1-deoxy-D-xylulose-5-phosphate reductoisomerase (see Example III and Figure
5).
[0126] The invention further provides a method for producing terephthalate, comprising culturing
a non-naturally occurring microbial organism containing a terephthalate pathways under
conditions and for a sufficient period of time to produce terephthalate. Such a terephthalate
pathway can comprise at least one exogenous nucleic acid encoding a terephthalate
pathway enzyme expressed in a sufficient amount to produce terephthalate, the terephthalate
pathway comprising
p-toluate methyl-monooxygenase reductase; 4-carboxybenzyl alcohol dehydrogenase; and/or
4-carboxybenzyl aldehyde dehydrogenase. Such a microbial organism can further comprise
a
p-toluate pathway, wherein the
p-toluate pathway comprises 2-dehydro-3-deoxyphosphoheptonate synthase; 3-dehydroquinate
synthase; 3-dehydroquinate dehydratase; shikimate dehydrogenase; shikimate kinase;
3-phosphoshikimate-2-carboxyvinyltransferase; chorismate synthase; and/or chorismate
lyase (see Examples IV and V and Figures 6 and 7). In another embodiment, the non-naturally
occurring microbial organism can further comprise a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate
pathway (see Example III and Figure 5). In some embodiments, the present invention
provides a method for producing toluene, comprising culturing a non-naturally occurring
microbial organism having a toluene pathway comprising at least one exogenous nucleic
acid encoding a toluene pathway enzyme expressed in a sufficient amount to produce
toluene. The toluene pathway can be selected from a set of pathway enzymes selected
from: a)
p-toluate decarboxylase; b)
p-toluate reductase and
p-methylbenzaldehyde decarbonylase; c)
p-toluate kinase, (
p-methylbenzoyloxy)phosphonate reductase, and
p-methylbenzaldehyde decarbonylase; d) (
p-methylbenzoyl-CoA synthetase, transferase and/or hydrolase), phosphotrans-
p-methylbenzoylase, (
p-methylbenzoyloxy)phosphonate reductase, and
p-methylbenzaldehyde decarbonylase; and e) (
p-methylbenzoyl-CoA synthetase, transferase and/or hydrolase),
p-methylbenzoyl-CoA reductase and
p-methylbenzaldehyde decarbonylase. The non-naturally occurring microbial organism
can be cultured under conditions and for a sufficient period of time to produce toluene.
In a particular embodiment, the invention provides a non-naturally occurring microbial
organism and methods of use, in which the microbial organism contains
p-toluate, terephthalate or toluene, and (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate
pathways.
[0127] In some embodiments, the present invention provides a method for producing (2-hydroxy-4-oxobutoxy)
phosphonate, comprising culturing a non-naturally occurring microbial organism having
a (2-hydroxy-4-oxobutoxy) phosphonate pathway comprising at least one exogenous nucleic
acid encoding a (2-hydroxy-4-oxobutoxy) phosphonate pathway enzyme expressed in a
sufficient amount to produce (2-hydroxy-4-oxobutoxy) phosphonate. The (2-hydroxy-4-oxobutoxy)
phosphonate pathway can include erythrose-4-phosphate dehydratase and (2,4-dioxobutoxy)phosphonate
reductase.
[0128] In some embodiments, the present invention provides a method for producing benzoate,
comprising culturing a non-naturally occurring microbial organism having a benzoate
pathway comprising at least one exogenous nucleic acid encoding a benzoate pathway
enzyme expressed in a sufficient amount to produce benzoate. The benzoate pathway
includes 2-dehydro-3-deoxyphosphoheptonate synthase; 3-dehydroquinate synthase; 3-dehydroquinate
dehydratase; shikimate dehydrogenase; shikimate kinase, 3-phosphoshikimate-2-carboxyvinyltransferase;
chorismate synthase; and chorismate lyase. The method includes culturing the non-naturally
occurring organism under conditions and for a sufficient period of time to produce
benzoate.
[0129] In some embodiments, the present invention provides a method for producing benzene,
comprising culturing the non-naturally occurring microbial organism having a benzene
pathway comprising at least one exogenous nucleic acid encoding a benzene pathway
enzyme expressed in a sufficient amount to produce benzene. The benzene pathway is
selected from a set of pathway enzymes selected from: a) benzoate decarboxylase; b)
benzoate reductase and benzaldehyde decarbonylase; c) benzoate kinase, (benzoyloxy)phosphonate
reductase, and benzaldehyde decarbonylase; d) (benzoyl-CoA synthetase, transferase
and/or hydrolase), phosphotransbenzoylase, (benzoyloxy)phosphonate reductase, and
benzaldehyde decarbonylase; and e) (benzoyl-CoA synthetase, transferase and/or hydrolase),
benzoyl-CoA reductase and benzaldehyde decarbonylase. The non-naturally occurring
microbial organism can be cultured under conditions and for a sufficient period of
time to produce benzene. In a particular embodiment, the invention provides a non-naturally
occurring microbial organism and methods of use, in which the microbial organism contains
(2-hydroxy-4-oxobutoxy) phosphonate, benzoate, and benzene pathways.
[0130] In some embodiments, the present invention provides a method for producing 2,4-pentadienoate
that includes culturing a non-naturally occurring microbial organism having a 2,4-pentadienoate
pathway. The pathway includes at least one exogenous nucleic acid encoding a 2,4-pentadienoate
pathway enzyme expressed in a sufficient amount to produce 2,4-pentadienoate, under
conditions and for a sufficient period of time to produce 2,4-pentadienoate. The 2,4-pentadienoate
pathway selected from A) i) a 4-hydroxy-2-oxovalerate aldolase, ii) a 4-hydroxy-2-oxovalerate
dehydratase, iii) a 2-oxopentenoate reductase, and iv) a 2-hydroxypentenoate dehydratase;
B) i) an AKP deaminase, ii) an acetylacrylate reductase, and iii) a 4-tiydroxypent-2-enoate
dehydratase; C) i) an AKP aminotransferase and/or dehydrogenase, ii) a 2,4-dioxopentanoate-2-reductase,
iii) a 2-hydroxy-4-oxopentanoate dehydratase, iv) an acetylacrylate reductase, and
v) a 4-hydroxypent-2-enoate dehydratase; D) i) an AKP aminotransferase and/or dehydrogenase,
ii) a 2,4-dioxopentanoate-4-reductase, iii) a 4-hydroxy-2-oxovalerate dehydratase,
iv) a 2-oxopentenoate reductase, and v) a 2-hydroxypentenoate dehydratase; and E)
i) an AKP reductase, ii) a 2-amino-4-hydroxypentanoate aminotransferase and/or dehydrogenase,
iii) a 4-hydroxy-2-oxovalerate dehydratase, iv) a 2-oxopentenoate reductase, and v)
a 2-hydroxypentenoate dehydratase.
[0131] In some embodiments, the methods of the invention utilize a non-naturally occurring
microbial organism having a 2,4-pentadienoate pathway that includes at least one exogenous
nucleic acid encoding a 2,4-pentadienoate pathway enzyme expressed in a sufficient
amount to produce 2,4-pentadienoate. The 2,4-pentadienoate pathway has a set of enzymes
selected from (A) 1) 4-hydroxy-2-oxovalerate aldolase, 2) 4-hydroxy-2-oxovalerate
reductase, 3) 2,4-dihydroxypentanoate 2-dehydratase, and 4) 4-hydroxypent-2-enoate
dehydratase, as shown in steps A, E, F, and G of Figure 12 and (B) 1) 4-hydroxy-2-oxovalerate
aldolase, 2) 4-hydroxy-2-oxovalerate reductase, 3) 2,4-dihydroxypentanoate 4-dehydratase
and 4) 2-hydroxypentenoate dehydratase, as shown in steps A, E, H, and D of Figure
12.
[0132] In some embodiments, the present methods of the invention utilize a non-naturally
occurring microbial organism having a 2,4-pentadienoate pathways that includes at
least one exogenous nucleic acid encoding a 2,4-pentadienoate pathway enzyme expressed
in a sufficient amount to produce 2,4-pentadienoate. The 2,4-pentadienoate pathway
has a set of enzymes selected from (A) 1) AKP aminotransferase and/or dehydrogenase,
2) 2,4-dioxopentanoate 2-reductase, 3) 2-hydroxy-4-oxopentanoate reductase, 4) 2,4-dihydroxypentanoate
2-dehydratase, and 5) 4-hydroxypent-2-enoate dehydratase, as shown, in steps E, H,
I, G, and D of Figure 13, and (B) 1) AKP aminotransferase and/or dehydrogenase, 2)
2,4-dioxopentanoate 2-reductase, 3) 2-hydroxy-4-oxopentanoate reductase, along with
4) 2,4-dihydroxypetanoate-2-dehydratase and 5) 4-hydroxypent-2-enoate dehydratase
or 4) 2,4-dihydroxypentanoate-4-dehydratase and 5) 2-hydroxypentenoate dehydratase,
as shown in steps E, H, and I of Figure 13, along with steps F and G or H and D of
Figure 12, respectively. That is to say, the double dehydration of 2,4-dihydroxypentanoate
can be performed in any order.
[0133] In some embodiments, the methods of the invention utilize a non-naturally occurring
microbial organism having an AKP pathway that includes at least one exogenous nucleic
acid encoding an AKP pathway enzyme expressed in a sufficient amount to produce AKP.
The AKP pathway includes an ornithine 4,5-aminomutase and a 2,4-diaminopentanoate
4-aminotransferase and/or 4-dehydrogenase, as shown in steps M and N of Figure 13.
In some embodiments, the microbial organism having an AKP pathway includes two exogenous
enzymes encoding an ornithine 4,5-aminomutase and a 2,4-diaminopentanoate 4-aminotransferase
or 2,4-diaminopentanoate 4-dehydrogenase. In some embodiments, this AKP pathway can
be added to any of the aforementioned 2,4-pentadienoate pathway and as indicated in
Figure 13. Alternatively, AKP can be accessed from alanine by addition of an AKP thiolase,
as shown, in step A of Figure 13, and fed into the various 2,4-pentadienoate pathways
described herein and shown in Figure 13, along with Figure 12.
[0134] In some embodiments, the methods of the invention utilize a non-naturally occurring
microbial organism having a 2,4-pentadienoate pathway that includes at least one exogenous
nucleic acid encoding a 2,4-pentadienoate pathway enzyme expressed in a sufficient
amount to produce 2,4-pentadienoate. The 2,4-pentadienoate pathway has a set of enzymes
selected from (A) 1) ornithine 2,3-aminomutase, 2) 3,5-diaminopentanoate deaminase,
and 3) 5-aminopent-2-enoate deaminase, as shown in steps A-C of Figure 14, (B) 1)
ornithine 2,3-aminomutase, 2) 3,5-diaminopentanoate deaminase, 3) 5-aminopent-2-enoate
aminotransferase and/or dehydrogenase, 4) 5-oxopent-2-enoate reductase, and 5) 5-hydroxypent-2-enoate
dehydratase, as shown in steps A, B, H, F, and G of Figure 14, (C) 1) ornithine 2,3-aminomutase,
2) 3,5-diaminopentanoate aminotransferase and/or dehydrogenase, 3) 3-amino-5-oxopentanoate
deaminase, 4) 5-oxopent-2-enoate reductase, and 5) 5-hydroxypent-2-enoate dehydratase
as shown in steps A, D, E, F, and G of Figure 14, and (D) 1) ornithine 2,3-aminomutase,
2) 3,5-diaminopentanoate aminotransferase and/or dehydrogenase, 3) 3-amino-5-oxopentanoate
reductase, and 4) 3-amino-5-hydroxypentanoate deaminase, and 5) 5-hydroxypent-2-enoate
dehydratase as shown in steps A, D, I, J, and G of Figure 14.
[0135] In some embodiments, the methods of the invention utilize a non-naturally occurring
microbial organism having a 2,4-pentadienoate pathway that includes at least one exogenous
nucleic acid encoding a 2,4-pentadienoate pathway enzyme expressed in a sufficient
amount to produce 2,4-pentadienoate. The 2,4-pentadienoate pathway has a set of enzymes
selected from any of the numerous pathway shown in Figure 15 starting from 3-HP-CoA
or acryloyl-CoA.
[0136] Exemplary pathways from 3-HP include the following enzyme sets (A) 1) 3-hydroxypropanoyl-CoA
acetyltransferase, 2) 3-oxo-5-hydroxypentanoyl-CoA reductase, 3) 3,5-dihydroxypentanoyl-CoA
dehydratase, 4) 5-hvdroxypent-2-enoyl-CoA dehydratase, and 5) pent-2,4-dienoyl-CoA
synthetase, transferase and/or hydrolase, as shown in steps A-E of Figure 15, and
(B) 1) 3-hydroxypropanoyl-CoA acetyl transferase, 2) 3-oxy-5-hydroxypentanoyl-CoA
synthetase, transferase and/or hydrolase, 3) 3-oxy-5-hydroxypentanoate reductase,
4) 3,5-dihydroxypentanoate dehydratase, and 5 5-hydroxypent-2-enoate dehydratase,
as shown in steps A, F, I, J, and Q of Figure 15. One skilled in the art will recognize
that enzyme sets defining pathways (A) and (B) from 3-HP-CoA can be intermingled via
reversible enzymes 3,5-hydroxypentanoyl-CoA synthetase, transferase, and/or hydrolase,
as shown by step G in Figure 15, and 5-hydroxypent-2-enoyl-CoA synthetase, transferase
and/or hydrolase, as shown by step H in Figure 15. Thus, a 3-HP-CoA to 2,4-pentadienoate
pathway can include the enzymes in steps A, B, G, J, and Q, or steps A, B, C, H, and
Q, or steps A, B, G, J, H, D, and E, or steps A, F, I, G, C, D, and E, or steps, A,
F, I, G, C, H, and Q, or steps A, F, I, J, H, D, and E, each shown in Figure 15.
[0137] Exemplary pathway from acryloyl-CoA include the following enzyme sets (C) 1) acryloyl-CoA
acetyltransferase, 2) 3-oxopent-4-enoyl-CoA synthetase, transferase and/or hydrolase,
3) 3-oxopent-4-enoate reductase, 4) 3-hydroxypent-4-enoate dehydratase, as shown in
steps M, O, P, and S in Figure 15 and (D), 1) acryloyl-CoA acetyltransferase, 2) 3-oxopent-4-enoyl-CoA
reductase, 3) 3-hydroxypent-4-enoyl-CoA dehydratase, and 4) pent-2,4-dienoyl-CoA synthetase,
transferase and/or hydrolase, as shown in steps M, N, R, E. One skilled in the art
will recognize that enzyme sets defining pathways (A) and (B) from 3-HP-CoA and (C)
and (D) from acryloyl-CoA can be intermingled via reversible enzymes 3-hydroxypropanoyl-CoA
dehydratase, as shown in step K of Figure 15, and 3-oxo-5-hydroxypentanoyl-CoA dehydratase,
as shown in step L of Figure 15. Thus, step K can be added to any of the enumerated
pathways from acryloyl-CoA to 2,4-pentadienoate providing 2,4-pentadienoate pathways
such as steps K, M, N, R, and E or steps K, M, O, P, and S. Step K can also be used
a shuttle alternative to step A to provide 3-oxo-5-hydroxypentanoyl-CoA from. 3-HP-CoA
via steps K, M, and L. Thus, any of the aforementioned pathways utilizing the enzyme
of step A can utilize the enzymes of steps K, M, and L, in its place. The save 3-oxo-5-hydroxypentanoyl-CoA
intermediate can be accessed from acryloyl-CoA by pathway via the enzymes of steps
K and A or M and L of Figure 15. Thus, acryloyl-CoA can be used to access all the
enumerated pathways that would be accessible from 3-HP-CoA. Thus, for example, an
acryloyl-CoA to 2,4-pentadienoate pathway can include enzymes from steps K, A, B,
C, D, and E, or steps K, A, F, I, J and Q, or steps K, A, B, G, J, and Q, or steps
K, A, B, G, J, H, D, and E, or steps K, A, B, C, H, and Q, or steps K, A, F, I, G,
C, D, and E, or steps K, A, F, I, G, C, H, Q, or steps K, A, F, I, J, H, D and E,
or steps M, L, B, C, D, and E, or steps M, L, F, I, J and Q, or steps M, L, B, G,
J, and Q, or steps M, L, B, G, J, H, D, and E, or steps M, L, B, C, H, and Q, or steps
M, L, F, I, G, C, D, and E, or steps M, L, F, I, G, C, H, Q, or steps M, L, F, I,
J, H, D and E, all as shown in Figure 15. Similarly, 3-HP-CoA can feed into the enumerated
acryloyl-CoA pathways via intermediate 3-oxopent-4-enoyl-CoA using the enzyme of step
L. Thus, a 3-HP-CoA to 2,4-pentadienoate pathway can include enzymes from steps A,
L, N, R, and E or steps A, L, O, P, and S, each pathway being shown in Figure 15.
[0138] In some embodiments, non-naturally occurring microbial organism used in methods of
the invention can include two exogenous nucleic acids each encoding a 2,4-pentadienoate
pathways enzyme. In some embodiments, non-naturally occurring microbial organism of
the invention can include three exogenous nucleic acids each encoding a 2,4-pentadienoate
pathways enzyme. For example, the non-naturally occurring microbial organism of the
invention can include three exogenous nucleic acids encoding i) an AKP deaminase,
ii) an acetylacrylate reductase, and iii) a 4-hydroxypent-2-enoate dehydratase, thus
providing an alanine or ornithine accessible pathway to 2,4-pentadienoate via AKP.
One skilled in the art will recognize that this is merely exemplary and that three
exogenous nucleic acids can be the basis of any 2,4-pentadienoate-producing non-naturally
occurring organism in any of the enumerated pathways of Figure 12-15.
[0139] In some embodiments, the non-naturalty occurring microbial organism used in methods
of the invention microbial can include any four erogenous nucleic acids each encoding
a 2,4-pentadienoate pathway enzyme. For example, a non-naturally occurring microbial
organism can include four exogenous nucleic acids encoding i) a 4-hydroxy-2-oxovalerate
aldolase, ii) a 4-hydroxy-2-oxovalerate dehydratase, iii) a 2-oxopentenoate reductase,
and iv) a 2-hydroxypentenoate dehydratase, thus defining a complete pathway from pyruvate
to 2,4-pentadienoate, as shown in Figure 12. One skilled in the art will recognize
that this is merely exemplary and that four exogenous nucleic acids can be the basis
of any 2,4-pentadienoate-producing non-naturally occurring organism in any of then
enumerated pathways of Figure 12-15.
[0140] In still further embodiments, the non-naturally occurring microbial organism used
in methods of the invention can include five exogenous nucleic acids each encoding
a 2,4-pentadienoate pathway enzyme. Exemplary non-naturally occurring microbial organism
of the invention having five exogenous nucleic acids can include enzymes encoding
(A) i) an AKP aminotransferase and/or dehydrogenase, ii) a 2,4-dioxopentanoate-2-reductase,
iii) a 2-hydroxy-4-oxopentanoate dehydratase, iv) an acetylacrylate reductase, and
v) a 4-hydroxypent-2-enoate dehydratase, as shown in steps E, H, F, C, and D in Figure
13, or (B) i) an AKP aminotransferase and/or dehydrogenase, ii) a 2,4-dioxopentanoate-4-reductase,
iii) a 4-hydroxy-2-oxovalerate dehydratase, iv) a 2-oxopentenoate reductase, and v)
a 2-hydroxypentenoate dehydratase, as shown in steps E and K of Figure 13, along with
steps B, C, and D of Figure 12, or i) an AKP reductase, ii) a 2-amino-4-hydroxypentanoate
aminotransferase and/or dehydrogenase, iii) a 4-hydroxy-2-oxovaterate dehydratase,
iv) a 2-oxopentenoate reductase, and v) a 2-hydroxypentenoate dehydratase, as shown
in steps J and L of Figure 13, along with steps B, C, and D of Figure 12. One skilled
in the art will recognize that this is merely exemplary and that five exogenous nucleic
acids can be the basis of any 2,4-pentadienoate-producing non-naturally occurring
organism in any of the enumerated pathways of Figure 12-15. Thus, in some embodiments
two, three, four, five, six, up to all of the enzymes in a 2,4-pentadienoate pathway
can be provided insertion of exogenous nucleic acids. In some embodiments, the non-naturally
occurring microbial organism of the invention has at least one exogenous nucleic acid
is a heterologous nucleic acid. Moreover, in some embodiments, the methods employing
non-naturally occurring microbial organism of the invention can utilize a substantially
anaerobic culture medium.
[0141] In some embodiments, the non-naturally occurring microbial organism used in methods
of the invention can further includes a 2,4-pentadienoate decarboxylase expressed
in a sufficient amount, to produce 1,3-butadiene by conversion of 2,4-pentadienoate
to 1,3-butadiene. Thus, any 2,4-pentadienoate pathway of Figure 12 can form the basis
of further production of 1,3 butadiene, as indicated by the conversion of cis or trans
2,4-pentadienoate to 1,3-butadiene in Figure 4.
[0142] In some embodiments, the present invention provide a method for producing 2,4-pentadienoate,
comprising culturing a non-naturally occurring microbial organism according to the
aforementioned pathways described herein, under conditions and for a sufficient period
of time to produce 2,4-pentadienoate. In some such embodiments, the microbial organism
includes two, three, four, five, six, seven, or eight exogenous nucleic acids each
encoding a 2,4-pentadienoate pathway enzyme. In some such embodiments, at least one
exogenous nucleic acid is a heterologous nucleic acid. In some such embodiments thenon-naturally
occurring microbial organism is in a substantially anaerobic culture medium.
[0143] In some embodiments, the present invention provides a method for producing 1,3-butadiene
that includes culturing a non-naturally occurring microbial organism according to
the aforementioned pathways described herein, under conditions and for a sufficient
period of time to produce 1,3-butadiene. In some such embodiments, the microbial organism
includes two, three, four, five, six, seven, or eight exogenous nucleic acids each
encoding a 1,3-butadiene pathway enzyme. In some such embodiments, at least one exogenous
nucleic acid is a heterologous nucleic acid. In some such embodiments, the non-naturally
occurring microbial organism is in a substantially anaerobic culture medium.
[0144] In some embodiments, the present invention provides a method for producing 3-butene-1-ol
which includes culturing a non-naturally occurring microbial organism according to
the aforementioned, pathway described herein, under conditions and for a sufficient
period of time to produce 3-butene-1-ol. In some such embodiments, the microbial organism
includes two, three, four, five, six, or seven exogenous nucleic acids each encoding
a 3-butene-1-ol pathway enzyme. In some such embodiments, at least one exogenous nucleic
acid is a heterologous nucleic acid. In some such embodiments, the non-naturally occurring
microbial organism is in a substantially anaerobic culture medium. In some such embodiments,
methods of the invention further include the chemical dehydration of 3-butene-1-ol
to provide 1,3-butadiene.
[0145] 3-butene-1-ol can be chemically dehydrated with formation of 1,3-butadiene, starting
with pure 3-butene-1-ol isolated from the fermentation solution or starting with an
aqueous or organic solutions of 3- butene-1-ol, isolated in work up of the fermentation
solution. Such solutions of 3- butene-1-ol can also be concentrated before the dehydration
step, for example by means of distillation, optionally in the presence of suitable
entrainer.
[0146] The dehydration reaction can be carried out in liquid phase or in the gas phase.
The dehydration reaction can be carried out in the presence of a catalyst, the nature
of the catalyst employed depending on whether a gas-phase or a liquid-phase reaction
is carried out.
[0147] Suitable dehydration catalysts include both acidic catalysts and alkaline catalysts.
Acidic catalysts, in particular can exhibits a decreased tendency to form oligomers.
The dehydration catalyst can be employed as a homogeneous catalyst, a heterogeneous
catalyst, or combinations thereof. Heterogeneous catalysts can be used in conjunction
with a suitable support material. Such a support can itself be acidic or alkaline
and provide the acidic or alkaline dehydration catalyst or a catalyst can be applied
to an inert support.
[0148] Suitable supports which serve as, dehydration catalysts include natural or synthetic
silicates such as mordenite, montmorillonite, acidic zeolites; supports which are
coated with monobasic, dibasic or polybasic inorganic acids, such as phosphoric acid,
or with acidic salts of inorganic acids, such as oxides or silicates, for example
Al
2O
3, TiO
2; oxides and mixed oxides such as γ-Al
2O
3 and ZnO-Al
2O
3 mixed oxides of heteropolyacids. Alkaline substances which act both as dehydration
catalyst and as a support a support material include alkali, alkaline earth, lanthanum,
lanthoids or a combinations thereof as their oxides. A further class of materials
that can effect dehydration are ion exchangers which can be used in either alkaline
or acidic form.
[0149] Suitable homogeneous dehydration catalysts include inorganic acids, such as phosphorus-containing
acids such as phosphoric acid. Inorganic acids can be immobilized on the support material
by immersion or impregnation.
[0150] In some embodiments, dehydration reaction is carried out in the gas phase using conventional
apparatuses known in the art, for example tubular reactors, shell-and-tube heat exchangers
and reactors which comprise thermoplates as heat exchangers. In some embodiments,
gas-phase dehydration can utilize isolated 3- butene-1-ol or solutions of butene-1-ol,
the butene-1-ol being introduced into a reactor with fixed-bed catalysts. Thermal
dehydration in the liquid phase can be carried out in a temperature range of between
200 °C and 350 °C, and in some embodiments between 250 and 300° C.
[0151] Suitable purification and/or assays to test for the production of toluene, benzene,
p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate,
benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol or 1,3-butadiene can be performed
using well known methods. Suitable replicates such as triplicate cultures can be grown
for each engineered strain to be tested. For example, product, and byproduct formation
in the engineered production host can be monitored. The final product and intermediate,
and other organic compounds, can be analyzed by methods such as HPLC (High Performance
Liquid Chromatography), GC-MS (Gas Chromatography-Mass Spectroscopy) and LC-MS (Liquid
Chromatography-Mass Spectroscopy) or other suitable analytical methods using routine
procedures well known in the art. The release of product in the fermentation broth
can also be tested with the culture supernatant. Byproducts and residual glucose can
be quantified by HPLC using, for example, a refractive index defector for glucose
and alcohols, and a UV detector for organic acids (
Lin et al., Biotechnol. Bioeng. 90:775-779 (2005)), or other suitable assay and detection methods well known in the art. The individual
enzyme or protein activities from the exogenous DNA sequences can also be assayed
using methods well known in the art. For example, the activity of phenylpyruvate decarboxylase
can be measured using a coupled photometric assay with alcohol dehydrogenase as an
auxiliary enzyme as described by Weiss et al (
Weiss et al., Biochem, 27:2197-2205 (1988). NADH- and NADPH-dependent enzymes such as acetophenone reductase can be followed
spectrophotometrically at 340 nm as described by Schlieben et al (
Schlieben et al, J.Mol. Bid., 349:801-813 (2005)). For typical hydrocarbon assay methods, see
Manual on Hydrocarbon Analysis (ASTM Manula Series, A.W. Drews, ed., 6th edition,
1998, American Society for Testing and Materials, Baltimore, Maryland. P-toluate methyl-monooxygenase activity can be assayed by incubating purified
enzyme with NADH, FeSO
4 and the p-toluate substrate in a water bath, stopping the reaction by precipitation
of the proteins, and analysis of the products in the supernatant by HPLC (
Locher et al., J. Bacteriol. 173:3741-3748 (1991)).
[0152] The toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate,
(2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol)
or 1,3-butadiene can be separated from other components in the culture using a variety
of methods well known in the art. Such separation methods include, for exampte, extraction
procedures as well as methods that include continuous liquid-liquid extraction, pervaporation,
membrane filtration, membrane separation, reverse osmosis, electrodialysis, distillation,
crystallization, centrifugation, extractive filtration, ion exchange chromatography,
exclusion chromatography, absorption chromatography, and ultrafiltration. All of the
above methods are well known in the art.
[0153] Any of the non-naturally occurring microbial organisms described herein can be cultured
to produce and/or secrete the biosynthetic products of the invention. For example,
the toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate,
(2-hydroxy-4-oxobatoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol
or 1,3-butadiene producers can be cultured for the biosynthetic production of toluene,
benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate,
benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol or 1,3-butadiene.
[0154] For the production of toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate,
(2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol
or 1,3-butadiene, the recombinant strains are cultured in a medium with carbon source
and other essential nutrients. It is sometimes desirable and can be highly desirable
to maintain anaerobic conditions in the fermenter to reduce the cost of the overall
process. Such conditions can be obtained, for example, by first sparging the medium
with nitrogen and then sealing the flasks with a septum and crimp-cap. For strains
where growth is not observed anaerobically, microaerobic or substantially anaerobic
conditions can be applied by perforating the septum with a small hole for limited
aeration. Exemplary anaerobic conditions have been described previously and are well-known
in the art. Exemplary aerobic and anaerobic conditions are described, for example,
in United States publication
2009/0047719, filed August 10, 2007. Fermentations can be performed in a batch, fed-batch or continuous manner, as disclosed
herein.
[0155] If desired, the pH of the medium can be maintained at a desired pH, in particular
neutral pH, such as a pH of around 7 by addition of a base, such as NaOH or other
bases, or acid, as needed to maintain the culture medium, at a desirable pH. The growth
rate can be determined by measuring optical density using a spectrophotometer (600
nm), and the glucose uptake rate by monitoring carbon source depletion over time.
[0156] The growth medium can include, for example, any carbohydrate source which can supply
a source of carbon to the non-naturally occurring microorganism. Such sources include,
for example, sugars such as glucose, xylose, arabinose, galactose, mannose, fructose,
sucrose and starch. Other sources of carbohydrate include, for example, renewable
feedstocks and biomass. Exemplary types of biomasses that can be used as feedstocks
in the methods of the invention include cellulosic biomass, hemicellulosic biomass
and lignin feedstocks or portions of feedstocks. Such biomass feedstocks contain,
for example, carbohydrate substrates useful as carbon sources such as glucose, xylose,
arabinose, galactose, mannose, fructose and starch. Given the teachings and guidance
provided herein, those skilled in the art will understand that renewable feedstocks
and biomass other than those exemplified above also can be used for culturing the
microbial organisms of the invention for the production of toluene, benzene, p-toluate,
terephthatate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate,
benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol or 1,3-butadiene.
[0157] In addition to renewable feedstocks such as those exemplified above, the toluene,
benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate,
benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene microbial organisms
of the invention also can be modified for growth on syngas as its source of carbon.
In this specific embodiment, one or more proteins or enzymes are expressed in the
toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate,
(2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol
or 1,3-butadiene producing organisms to provide a metabolic pathway for utilization
of syngas or other gaseous carbon source.
[0158] Synthesis gas, also known as syngas or producer gas, is the major product of gasification
of coal and of carbonaceous materials such as biomass materials, including agricultural,
crops and residues. Syngas is a mixture primarily of H
2 and CO and can be obtained from the gasification of any organic feedstock, including
but not limited to coal, coal oil, natural gas, biomass, and waste organic matter.
Gasification is generally carried out under a high fuel to oxygen ratio. Although
largely H
2 and CO, syngas can also include CO
2 and other gases in smaller quantities. Thus, synthesis gas provides a cost effective
source of gaseous carbon such as CO and, additionally, CO
2.
[0159] The Wood-Ljungdahl pathway catalyzes the conversion of CO and H
2 to acetyl-CoA and other products such as acetate. Organisms capable of utilizing
CO and syngas also generally have the capability of utilizing CO
2 and CO
2/H
2 mixtures through the same basic set of enzymes and transformations encompassed by
the Wood-Ljungdahl pathway. H
2-dependent conversion of CO
2 to acetate by microorganisms was recognized long before it was revealed that CO also
could be used by the same organisms and that the same pathways were involved. Many
acetogens have been shown to grow in the presence of CO
2 and produce compounds such as acetate as long as hydrogen is present to supply the
necessary reducing equivalents (see for example,
Drake, Acetogenesis, pp. 3-60 Chapman and Hall, New York, (1994)). This can be summarized by the following equation:
2 CO
2 + 4 H
2 + n ADP + n Pi → CH
3COOH + 2 H
2O + n ATP
[0160] Hence, non-naturally occurring microorganisms possessing the Wood-Ljungdahl pathway
can utilize CO
2 and H
2 mixtures as well for the production of acetyl-CoA and other desired products.
[0161] The Wood-Ljungdahl pathway is well known in the art and consists of 12 reactions
which can be separated into two branches: (1) methyl branch and (2) carbonyl branch.
The methyl branch converts syngas to methyl-tetrahydrofolate (methyl-THF) whereas
the carbonyl branch converts methyl-THF to acetyl-CoA. The reactions in the methyl
branch are catalyzed in order by the following enzymes or proteins: ferredoxin oxidoreductase,
formate dehydrogenase, formyltetrahydrofolate synthetase, methenyltetrahydrofolate
cyclodehydratase, methylenetetrahydrofolate dehydrogenase and methylenetetrahydrofolate
reductase. The reactions in the carbonyl branch are catalyzed in order by the following
enzymes or proteins: methyltetrahydrofolate:corrinoid protein methyltransferase (for
example, AcsE), corrinoid iron-sulfur protein, nickel-protein assembly protein (for
example, AcsF), ferredoxin, acetyl-CoA synthase, carbon monoxide dehydrogenase and
nickel-protein assembly protein (for example, CooC). Following the teachings and guidance
provided herein for introducing a sufficient number of encoding nucleic acids to generate
a toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate,
(2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol
or 1,3-butadiene pathway, those skilled in the art will understand that the same engineering
design also can be performed with respect to introducing at least the nucleic acids
encoding the Wood-Ljungdahl enzymes or proteins absent in the host organism. Therefore,
introduction of one or more encoding nucleic acids into the microbial organisms of
the invention such that the modified organism contains the complete Wood-Ljungdahl
pathway will confer syngas utilization ability.
[0162] Additionally, the reductive (reverse) tricarboxylic acid cycle coupled with carbon
monoxide dehydrogenase and/or hydrogenase activities can also be used for the conversion
of CO, CO
2 and/or H
2 to acetyl-CoA and other products such as acetate. Organisms capable of fixing carbon
via the reductive TCA pathway can utilize one or more of the following enzymes: ATP
citrate-lyase, citrate lyase, aconitase, isocitrate dehydrogenase, alpha-ketoglutarate:ferredoxin
oxidoreductase, succinyl-CoA synthetase, succinyl-CoA transferase, fumarate reductase,
fumarase, malate dehydrogenase, NAD(P)H:ferredoxin oxidoreductase, carbon monoxide
dehydrogenase, and hydrogenase. Specifically, the reducing equivalents extracted from
CO and/or H
2 by carbon monoxide dehydrogenase and hydrogenase are utilized to fix CO
2 via the reductive TCA cycle into acetyl-CoA or acetate. Acetate can be converted
to acetyl-CoA by enzymes such as acetyl-CoA transferase, acetate kinase/phosphotransacetylase,
and acetyl-CoA synthetase. Acetyl-CoA can be converted to the toluene, benzene, p-toluate,
terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)ptiosphonate,
benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene precursors, glyceraldehyde-3-phosphate,
phosphoenolpyruvate, and pyruvate, by pyruvate:ferredoxin oxidoreductase and the enzymes
of gluconeogenesis. Following the teachings and guidance provided herein for introducing
a sufficient number of encoding nucleic acids to generate a toluene, benzene, p-toluate,
terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate,
benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol or 1,3-butadiene pathway, those
skilled in the art will understand that the same engineering design also can be performed
with respect to introducing at least the nucleic acids encoding the reductive TCA
pathway enzymes or proteins absent in the host organism. Therefore, introduction of
one or more encoding nucleic acids into the microbial organisms of the invention such
that the modified organism contains the complete reductive TCA pathway will confer
syngas utilization ability.
[0163] In some embodiments, the invention provides a non-naturally occurring microbial organism,
comprising a microbial organism having a 1,3-butadiene pathway comprising at least
one exogenous nucleic acid encoding a 1,3-butadiene pathway enzyme expressed in a
sufficient amount to produce 1,3-butadiene, said 1,3-butadiene pathway selected from:
(A) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA transferase, a 3-oxoadipyl-CoA
synthetase or a 3-oxoadipyl-CoA hydrolase; a 3-oxoadipate dehydrogenase; a 2-fumarylacetate
decarboxylase; a 3-oxopent-4-enoate reductase; and a 3-hydroxypent-4-enoate decarboxylase;
(B) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA transferase, a 3-oxoadipyl-CoA
synthetase or a 3-oxoadipyl-CoA hydrolase; a 3-oxoadipate dehydrogenase; a 2-fumarylacetate
decarboxylase; a 3-oxopent-4-enoate reductase; a 3-hydroxypent-4-enoate dehydratase;
and a 2,4-pentadicnoate decarboxylase; (C) a succinyl-CoA:acetyl-CoA acyltransferase;
a 3-oxoadipyl-CoA transferase, a 3-oxoadipyl-CoA synthetase or a 3-oxoadipyl-CoA hydrolase;
a 3-oxoadipate dehydrogenase; a 2-fumarylacetate reductase; a 3-hydroxyhex-4-enedioate
decarboxylase; and a 3-hydroxypent-4-enoate decarboxylase; (D) a succinyl-CoA:acetyl-CoA
acyltransferase; a 3-oxoadipyl-CoA transferase, a 3-oxoadipyl-CoA synthetase or a
3-oxoadipyl-CoA hydrolase;a 3-oxoadipate dehydrogenase; a 2-fumarylacetate reductase;
a 3-hydroxyhex-4-enedioate decarboxylase; a 3-hydroxypent-4-enoate dehydratase; and
a 2,4-pentadienoate decarboxylase; (E) a succinyl-CoA:acetyl-CoA acyltransferase;
a 3-oxoadipyl-CoA transferase, a 3-oxoadipyl-CoA synthetase or a 3-oxoadipyl-CoA hydrolase;
a 3-oxoadipate reductase; a 3-hydroxyadipate dehydrogenase; a 3-hydroxyhex-4-enedioate
decarboxylase; and a 3-hydroxypent-4-enoate decarboxylase; (F) a succinyl-CoA:acetyl-CoA
acyltransferase; a 3-oxoadipyl-CoA transferase, a 3-oxoadipyl-CoA synthetase or a
3-oxoadipyl-CoA hydrolase; a 3-oxoadipate reductase; a 3-hydroxyadipate dehydrogenase;
a 3-hydroxyhex-4-enedioate decarboxylase; a 3-hydroxypent-4-enoate dehydratase; and
a 2,4-pentadienoate decarboxylase; (G) a succinyl-CoA:acetyl-CoA acyltransferase;
a 3-oxoadipyl-CoA reductase; a 3-hydroxyadipyl-CoA transferase, a 3-hydroxyadipyl-CoA
synthetase or a 3-hydroxyadipyl-CoA hydrotase; a 3-hydroxyadipate dehydrogenase; a
3-hydroxyhex-4-enedioate decarboxylase; and a 3-hydroxypent-4-enoate decarboxylase;
and (H) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA reductase; a
3-hydroxyadipyt-CoA transferase, a 3-hydroxyadipyl-CoA synthetase or a 3-hydroxyadipyl-CoA
hydrolase; a 3-hydroxyadipate dehydrogenase; a 3-hydroxyhex-4-enedioate decarboxylase;
a 3-hydroxypent-4-enoate dehydratase; and a 2,4-pentadienoate decarboxylase.
[0164] In some aspects, the non-naturally occurring microbial organism comprises two, three,
four, five, six or seven exogenous nucleic acids each encoding a, 1,3-butadiene pathway
enzyme. For example, the microbial organism can comprise exogenous nucleic acids encoding
each of the enzymes selected from: (A) a succinyl-CoA:acetyl-CoA acyltransferase;
a 3-oxoadipyl-CoA transferase, a 3-oxoadipyl-CoA synthetase or a 3-oxoadipyl-CoA hydrolase;
a 3-oxoadipate dehydrogenase; a 2-fumarylacetate decarboxylase; a 3-oxopent-4-enoate
reductase; and a 3-hydroxypent-4-enoate decarboxylase; (B) a succinyl-CoA:acetyl-CoA
acyltransferase; a 3-oxoadipyl-CoA transferase, a 3-oxoadipyl-CoA synthetase or a
3-oxoadipy)-CoA hydrolase; a 3-oxoadipate dehydrogenase; a 2-fumarylacetate decarboxylase;
a 3-oxopent-4-enoate reductase; a 3-hydroxypent-4-enoate dehydratase; and a 2,4-pentadienoate
decarboxylase; (C) a succinyl-CoA:acetyl-CoA acyltransferase, a 3-oxoadipyl-CoA transferase,
a 3-oxoadipyl-CoA synthetase or a 3-oxoadipyl-CoA hydrolase; a 3-oxoadipate dehydrogenase;
a 2-fumarylacetate reductase; a 3-hydroxyhex-4-enedioate decarboxylase; and a 3-hydroxypent-4-enoate
decarboxylase; (D) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA transferase,
a 3-oxoadipyl-CoA synthetase or a 3-oxoadipyl-CoA hydrolase; a 3-oxoadipate dehydrogenase;
a 2-fumarylacetate reductase; a 3-hydroxyhex-4-enedioate decarboxytase; a 3-hydroxypent-4-enoate
dehydratase; and a 2,4-pentadienoate decarboxylase; (E) a succinyl-CoA:acetyl-CoA
acyltransferase; a 3-oxoadipyl-CoA transferase, a 3-oxoadipyl-CoA synthetase or a
3-oxoadipyl-CoA hydrolase; a 3-oxoadipate reductase; a 3-hydroxyadipate dehydrogenase;
a 3-hydroxyhex-4-enedioate decarboxylase; and a 3-hydroxypent-4-enoate decarboxylase;
(F) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA transferase, a 3-oxoadipyl-CoA
synthetase or a 3-oxoadipyl-CoA hydrolase; a 3-oxoadipate reductase; a 3-hydroxyadipate
dehydrogenase; a 3-hydroxyhex-4-enedioate decarboxylase; a 3-hydroxypent-4-enoate
dehydratase; and a 2,4-pentadienoate decarboxylase; (G) a succinyl-CoA:acetyl-CoA
acyltransferase; a 3-oxoadipyl-CoA reductase; a 3-hydroxyadipyl-CoA transferase, a
3-hydroxyadipyl-CoA synthetase or a 3-hydroxyadipyl-CoA hydrolase; a 3-hydroxyadipate
dehydrogenase; a 3-hydroxyhex-4-enedioate decarboxylase; and a 3-hydroxypent-4-enoate
decarboxylase; and (H) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA
reductase; a 3-hydroxyadipyl-COA transferase, a, 3-hydroxyadipyl-CoA synthetase or
a 3-hydroxyadipyl-CoA hydrolase; a 3-hydroxyadipate dehydrogenase; a 3-hydroxyhex-4-enedioate
decarboxylase; a 3-hydroxypent-4-enoate dehydratase; and a 2,4-pentadienoate decarboxylase.
[0165] In some embodiments, the invention provides a non-naturally occurring microbial organism
as described above, wherein said microbial organism further comprises; (i) a reductive
TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA
pathway enzyme, wherein said at least one exogenous nucleic acid is selected from
an ATP-citrate lyase, a citrate lyase, a fumarate reductase, and an alpha-ketoglutarate;
ferredoxin oxidoreductase; (ii) a reductive TCA pathway comprising at least one exogenous
nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous
nucleic acid is selected from a pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate
carboxylase, a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H2 hydrogenase;
or (iii) at least one exogenous nucleic acid encodes an enzyme selected from a CO
dehydrogenase, an H2 hydrogenase, and combinations thereof. In some aspects of the
invention, the microbial organism comprising (i) further comprises an exogenous nucleic
acid encoding an enzyme selected from a pyruvate:ferredoxin oxidoreductase, an aconitase,
an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase,
a fumarase, a malate dehydrogenase, an acetate kinase, a phosphotransacetylase, an
acetyl-CoA synthetase, an NAD(P)H:ferredoxin oxidoreductase, ferredoxin, and combinations
thereof. In some aspects of the invention, the microbial organism comprising (ii)
further comprises an exogenous nucleic acid encoding an enzyme selected from an aconitase,
an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase,
a fumarase, a malate dehydrogenase, and combinations thereof.
[0166] In some embodiments, the invention provides a non-naturally occurring microbial organism
of as described above, wherein said microbial organism comprising (i) comprises four
exogenous nucleic acids encoding an ATP-citrate lyase, citrate lyase, a fumarate reductase,
and an alpha-ketoglutarate:ferredoxin oxidoreductase; wherein said microbial organism
comprising (ii) comprises five exogenous nucleic acids encoding a pyruvate:ferredoxin
oxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase,
a CO dehydrogenase, and an H
2 hydrogenase; or wherein said microbial organism comprising (iii) comprises two exogenous
nucleic acids encoding a CO dehydrogenase and an H
2 hydrogenase.
[0167] In some aspects, the invention provides that the non-naturally occurring microbial
organism as described herein, wherein said at least one exogenous nucleic acid is
a heterologous nucleic acid. In another aspect, the non-naturally occurring microbial
organism is in a substantially anaerobic culture medium. In some embodiments, the
invention provides a method for producing 1,3-butadiene, comprising culturing a non-naturally
occurring microbial organism as described herein under conditions and for a sufficient
period of time to produce 1,3-butadiene.
[0168] In some embodiments, the invention provides non-naturally occurring microbial organism,
comprising a microbial organism having a 2,4-pentadienoate pathway comprising at least
one exogenous nucleic acid encoding a 2,4-pentadienoate pathway enzyme expressed in
a sufficient amount to produce 2,4-pentadienoate, 2,4-pentadienoate pathway selected
from: (A) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA transferase,
a 3-oxoadipyl-CoA synthetase ora 3-oxoadipyl-CoA hydrolase; a 3-oxoadipate dehydrogenase;
a 2-fumarylacetate decarboxylase; a 3-oxopent-4-enoate reductase; and a 3-hydroxypent-4-enoate
dehydratase; (B) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA transferase,
a 3-oxoadipyl-CoA synthetase ora 3-oxoadipyl-CoA hydrolase; a 3-oxoadipate dehydrogenase;
a 2-fumarylacetate reductase; a 3-hydroxyhex-4-enedioate decarboxylase; and a 3-hydroxypent-4-enoate
dehydratase; (C) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA transferase,
a 3-oxoadipyl-CoA synthetase ora 3-oxoadipyl-CoA hydrolase; a 3-oxoadipate reductase;
a 3-hydroxyadipate dehydrogenase; a 3-hydroxyhex-4-enedioate decarboxylase; and a
3-hydroxypent-4-enoate dehydratase; and (D) a succinyl-CoA:acetyl-CoA acyltransferase;
a 3-oxoadipyl-CoA reductase; a 3-hydroxyadipyl-CoA transferase, a 3-hydroxyadipyl-CoA
synthetase or a 3-hydroxyadipyl-CoA hydrolase; a 3-hydroxyadipate dehydrogenase; a
3-hydroxyhex-4-enedioate decarboxylase; and a 3-hydroxypent-4-enoate dehydratase.
[0169] In some aspects, the microbial organism comprises two, three, four, five, or six
exogenous nucleic acids each encoding a 2,4-pentadienoate pathway enzyme. For example,
the microbial organism can comprise exogenous nucleic acids encoding each of the enzymes
selected from: (A) a succiny)-CoA:acetyl-CoA. acyltransferase; a 3-oxoadipyl-CoA transferase,
a 3-oxoadipyl-CoA synthetase ora 3-oxoadipyl-CoA hydrolase; a 3-oxoadipate dehydrogenase;
a 2-fumarylacetate decarboxylase; a 3-oxopent-4-enoate reductase; and a 3-hydroxypent-4-enoate
dehydratase; (B) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA transferase,
a 3-oxoadipyl-CoA synthetase ora 3-oxoadipyl-CoA hydrolase; a 3-oxoadipate dehydrogenase;
a 2-fumarylacetate reductase; a 3-hydroxyhex-4-enedioate decarboxylase; and a 3-hydroxypent-4-enoate
dehydratase; (C) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA transferase,
a 3-oxoadipyl-CoA synthetase ora 3-oxoadipyl-CoA hydrolase; a 3-oxoadipate reductase;
a 3-hydroxyadipate dehydrogenase; a 3-hydroxyhex-4-enedioate decarboxylase; and a
3-hydroxypent-4-enoate dehydratase; and (D) a succinyl-CoA:acetyl-CoA acyltransferase;
a 3-oxoadipyl-CoA reductase; a 3-hydroxyadipyl-CoA transferase, a 3-hydroxyadipyl-CoA
synthetase or a 3-hydroxyadipyl-CoA hydrolase; a 3-hydroxyadipate dehydrogenase; a
3-hydroxyhex-4-enedioate decarboxytase; and a 3-hydroxypent-4-enoate dehydratase.
[0170] In some embodiments, the invention provides a non-naturally occurring microbial organism,
as described above, wherein said microbial organism further comprises: (i) a reductive
TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA
pathway enzyme, wherein said at least one exogenous nucleic acid is selected from
an ATP-citrate lyase, a citrate lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin
oxidoreductase; (ii) a reductive TCA pathway comprising at least one exogenous nucleic
acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous
nucleic acid is selected from a pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate
carboxylase, a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H2 hydrogenase;
or (iii) at least one exogenous nucleic acid encodes an enzyme selected from a CO
dehydrogenase, an H2 hydrogenase, and combinations thereof. In some aspects, the microbial
organism comprising (i) further comprises an exogenous nucleic acid encoding an enzyme
selected from a pyruvate:ferredoxin oxidoreductase, an aconitase, an isocitrate dehydrogenase,
a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase,
an acetate a phosphotransacetylase, an acetyl-CoA synthetase, an NAD(P)H:ferredoxin
oxidoreductase, ferredoxin, and combinations thereof. In some aspects, the microbial
organism comprising (ii) further comprises an exogenous nucleic acid encoding an enzyme
selected from an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase,
a succinyl-CoA transferase, a fumarase, a malate and combinations thereof.
[0171] In some aspects, the non-naturally occurring microbial orgnaism comprising (i) comprises
four exogenous nucleic acids encoding an ATP-citrate lyase, citrate lyase, a fumarate
reductase, and an alpha-ketoglutarate:ferredoxin oxidoreductase; wherein said microbial
organism comprising (ii) comprises five exogenous nucleic acids encoding a pyruvate:ferredoxin
oxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase,
a CO dehydrogenase, and an H
2 hydrogenase; or wherein said microbial organism comprising (iii) comprises two exogenous
nucleic acids encoding a CO dehydrogenase and an H
2 hydrogenase.
[0172] In some aspects, the invention provides that the non-naturally occurring microbial
organism as disclosed above, wherein said at least one exogenous nucleic acid is a
heterologous nucleic acid. In some aspects, the non-naturally occurring microbial
organism is in a substantially anaerobic culture medium.
[0173] In some embodiments, the invention provides a method for producing 2,4-pentadienoate,
comprising culturing a non-naturally occurring microbial organism as described herein
under conditions and for a sufficient period of time to produce 2,4-pentadienoate.
[0174] In some embodiments, the invention provides a non-naturally occurring microbial organism,
comprising a microbial organism having a 1,3-butadiene pathway comprising at least
one exogenous nucleic acid encoding a 1,3-butadiene pathway enzyme expressed in a
sufficient amount to produce 1,3-butadiene, said 1,3-butadiene pathway selected from:
(A) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde
forming); a 3,5-dioxopentanoate reductase (aldehydes reducing); a 5-hydroxy-3-oxopentanoate
reductase; a 3,5-dihydroxypentanoate dehydratase; a 5-hydroxypent-2-enoate dehydratase;
and a 2,4-pentadiene decarboxylase; (B) a malonyl-CoA:acetyl-CoA acyltransferase;
a 3-oxoglutaryl-CoA reductase (aldehyde forming); a 3,5-dioxopentanoate reductase
(aldehyde reducing); a 5-hydroxy-3-oxopentanoate reductase; a 3,5-dihydroxypentanoate
dehydratase; a 5-hydroxypent-2-enoate decarboxylase; and a 3-butene-1-ol dehydratase
; (C) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde
forming; a 3,5-dioxopentanoate reductase (aldehyde reducing); a 5-hydroxy-3-oxopentanoate
reductase; a 3,5-dihydroxypentanoate decarboxylase; and a 3-butene-1-ol dehydratase;
(D) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (CoA reducing
and alcohol forming); a 5-hydroxy-3-oxopentanoate reductase; a 3,5-dihydroxypentanoate
dehydratase; a 5-hydroxypent-2-enoate dehydratase; and a 2,4-pentadiene decarboxylase;
(E) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (CoA reducing
and alcohol forming); a 5-hydroxy-3-oxopentanoate reductase; a 3,5-dihydroxypentanoate
dehydratase; a 5-hydroxypent-2-enoate decarboxylase; and a 3-butene-1-ol dehydratase;
(F) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (CoA reducing
and alcohol forming); a 5-hydroxy-3-oxopentanoate reductase; a 3,5-dihydroxypentanoate
decarboxylase; and a 3-butene-1-ol dehydratase; (G) a malonyl-CoA:acetyl-CoA acyltransferase;
a 3-oxoglutaryl-CoA reductase (aldehyde forming); a 3,5-dioxopentanoate reductase
(ketone reducing); a 3-hydroxy-5-oxopentanoate reductase; a 3,5-dihydroxypentanoate
dehydratase; a 5-hydroxypent-2-enoate dehydratase; and a 2,4-pentadiene decarboxylase;
(H) a malonyl-CoA:acctyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde
forming); a 3,5-dioxopentanoate reductase (ketone reducing); a 3-hydroxy-5-oxopentanoate
reductase; a 3,5-dihydroxypentanoate dehydratase; a 5-hydroxypent-2-enoate decarboxylase;
and a 3-butene-1-ol dehydratase; (I) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA
reductase (aldehyde forming); a 3,5-dioxopentanoate reductase (ketone reducing); a
3-hydroxy-5-oxopentanoate reductase; a 3,5-dihydroxypentanoate decarboxylase; and
a 3-butene-1-ol dehydratase; (J) a malony-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA
reductase (ketone-reducing); a 3-hydroxyglutaryl-CoA reductase (aldehyde forming);
a 3-hydroxy-5-oxopentanoate reductase; a 3,5-dihydroxypentanoate dehydratase; a 5-hydroxypent-2-enoate
dehydratase; and a 2,4-pentadiene decarboxylase; (K) a malonyl-CoA:acetyl-CoA acyltransferase;
a 3-oxoglutaryt-CoA reductase (ketone-reducing); a 3-hydroxyglutaryl-CoA reductase
(aldehyde forming); a 3-hydroxy-5-oxopentanoate reductase; a 3,5-dihydroxypentanoate
dehydratase; a 5-hydroxypent-2-enoate decarboxylase; and a 3-butene-1-ol dehydratase;
(L) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing);
a 3-hydroxyglutaryl-CoA reductase (aldehyde forming); a 3-hydroxy-5-oxopentanoate
reductase; a 3,5-dihydroxypentanoate decarboxylase; and a 3-butene-1-ol dehydratase;
(M) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing);
a 3-hydroxyglutaryl-CoA reductase (alcohol forming); a 3,5-dihydroxypentanoate dehydratase;
a 5-hydroxypent-2-enoate dehydratase; and a 2,4-pentadiene decarboxylase; (N) a malonyl-CoA:acetyl-CoA
acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing); a 3-hydroxyglutaryl-CoA
reductase (alcohol forming); a 3,5-dihydroxypentanoate dehydratase; a 5-hydroxypent-2-enoate
decarboxylase; and a 3-butene-1-ol dehydratase; and (O) a malonyl-CoA:acetyl-CoA acyltransferase;
a 3-oxoglutaryl-CoA reductase (ketone-reducing); a 3-hydroxyglutaryl-CoA reductase
(alcohol forming); a 3,5-dihydroxypentanoate decarboxylase; and a 3-butene-1-ol dehydratase.
[0175] In some embodiments, the invention provides a non-naturally occurring microbial organism
as described above, wherein said microbial organism comprises two, three, four, five,
six or seven exogenous nucleic acids each encoding a 1,3-butadiene pathway enzyme.
For example, in some aspects, the microbial organism comprises exogenous nucleic acids
encoding each of the enzymes selected from: (A) a malonyl-CoA:acetyl-CoA acyltransferase;
a 3-oxoglutaryl-CoA reductase (aldehyde forming); a 3,5-dioxopentanoate reductase
(aldehyde reducing); a 5-hydroxy-3-oxopentanoate reductase; a 3,5-dihydroxypentanoate
dehydratase; a 5-hydroxypent-2-enoate dehydratase; and a 2,4-pentadiene decarboxylase;
(B) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde
forming); a 3,5-dioxopentanoate reductase (aldehyde reducing); a 5-hydroxy-3-oxopentanoate
reductase; a 3,5-dihydroxypentanoate dehydratase; a 5-hydroxypent-2-enoate decarboxylase;
and a 3-butene-1-ol dehydratase; (C) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryt-CoA
reductase (aldehyde forming); a 3,5-dioxopentanoate reductase (aldehyde reducing);
a 5-hydroxy-3-oxopentanoate reductase; a 3,5-dihydroxypentanoate decarboxylase; and
a 3-butene-1-ol dehydratase; (D) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA
reductase (CoA reducing and alcohol forming); a 5-hydroxy-3-oxopentanoate reductase;
a 3,5-dihydroxypentanoate dehydratase; a 5-hydroxypent-2-enoate dehydratase; and a
2,4-pentadiene decarboxylase; (E) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA
reductase (CoA reducing and alcohol forming); a 5-hydroxy-3-oxopentanoate reductase;
a 3,5-dihydroxypentanoate dehydratase; a 5-hydroxypent-2-enoate decarboxylase; and
a 3-butene-1-ol dehydratase; (F) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA
reductase (CoA reducing and alcohol forming); a 5-hydroxy-3-oxopentanoate reductase;
a 3,5-dihydroxypentanoate decarboxylase; and a 3-butene-1-ol dehydratase; (G) a malonyl-CoA:acetyl-CoA
acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde forming); a 3,5-dioxopentanoate
reductase (ketone reducing); a 3-hydroxy-5-oxopentanoate reductase; a 3,5-dihydroxypentanoate
dehydratase; a 5-hydroxypent-2-enoate dehydratase; and a 2,4-pentadiene decarboxylase;
(H) a malony-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde
forming); a 3,5-dioxopentanoate reductase (ketone reducing); a 3-hydroxy-5-oxopentanoate
reductase; a 3,5-dihydroxypentanoate dehydratase; a 5-hydroxypent-2-enoate decarboxylase;
and a 3-butene-1-ol dehydratase; (I) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA
reductase (aldehyde forming); a 3,5-dioxopentanoate reductase (ketone reducing); a
3-hydroxy-5-oxopentanoate reductase; a 3,5-dihydroxypentanoate decarboxylase; and
a 3-butene-1-ol dehydratase; (J) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA
reductase (ketone-reducig); a 3-hydroxyglutaryl-CoA reductase (aldehyde forming);
a 3-hydroxy-5-oxopentanoate reductase; a 3,5-dihydroxypentanoate dehydratase; a 5-hydroxypent-2-enoate
dehydratase; and a 2,4-pentadiene decarboxylase; (K) a malonyl-CoA:acetyl-CoA acyltransferase;
a 3-oxoglutaryl-CoA reductase (ketone-reducing); a 3-hydroxyglutaryl-CoA reductase
(aldehyde forming); a 3-hydroxy-5-oxopentanoate reductase; a 3,5-dihydroxypentanoate
dehydratase
; a 5-hydroxypent-2-enoate decarboxylase; and a 3-butene-1-ol dehydratase; (L) a malonyl-CoA:acetyl-CoA
acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing); a 3-hydroxyglutaryl-CoA
reductase (aldehyde forming); a 3-hydroxy-5-oxopentanoate reductase; a 3,5-dihydroxypentanoate
decarboxylase; and a 3-butene-1-ol dehydratase; (M) a malonyl-CoA:acetyl-CoA acyltransferase;
a 3-oxoglutaryl-CoA reductase (ketone-reducing); a 3-hydroxyglutaryl-CoA reductase
(alcohol forming); a 3,5-dihydroxypentanoate dehydratase; a 5-hydroxypent-2-enoate
dehydratase; and a 2,4-pentadiene decarboxylase; (N) a malonyl-CoA:acetyl-CoA acyltransferase;
a 3-oxoglutaryl-CoA reductase (ketone-reducing); a 3-hydroxyglutaryl-CoA reductase
(alcohol forming); a 3,5-dihydroxypentanoate dehydratase; a 5-hydroxypent-2-enoate
decarboxylase; and a 3-butene-1-ol dehydratase; and (O) a malonyl-CoA:acetyl-CoA acyltransferase;
a 3-oxoglutaryl-CoA reductase (ketone-reducing); a 3-hydroxyglutaryl-CoA reductase
(alcohol forming); a 3,5-dihydroxypentanoate decarboxylase; and a 3-butene-1-ol dehydratase.
[0176] In some embodiments, the invention provides a non-naturally occurring microbial organism
as disclosed above, wherein said microbial organism further comprises: (i) a reductive
TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA
pathway enzyme, wherein said at least one exogenous nucleic acid is selected from
an ATP-citrate lyase, a citrate lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin
oxidoreductase; (ii) a reductive TCA pathway comprising at least one exogenous nucleic
acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous
nucleic acid is selected from a pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate
carboxylase, a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H2 hydrogenase;
or (iii) at least one exogenous nucleic acid encodes an enzyme selected from a CO
dehydrogenase, an H2 Hydrogenase, and combinations thereof. In some aspects, the non-naturally
occurring microbial organism comprising (i) further comprises an exogenous nucleic
acid encoding an enzyme selected from a pyruvate:ferredoxin oxidoreductase, an aconitase,
an isocitrate dehydrogenase, a succinyl-COA synthetase, a succinyl-CoA transferase,
a fumarase, a malate dehydrogenase, an acetate kinase, a phosphotransacetylase, an
acetyl-CoA synthetase, an NAD(P)H:ferredoxin oxidoreductase, ferredoxin, and combinations
thereof. In some aspects, the microbial organism comprising (ii) further comprises
an exogenous nucleic acid encoding an enzyme selected from an aconitase, an isocitrate
dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase,
a malate dehydrogenase, and combinations thereof. In some aspects, the microbial organism
comprising (i) comprises four exogenous nucleic acids encoding an ATP-citrate lyase,
citrate lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin oxidoreductase;
wherein said microbial organism comprising (ii) comprises five exogenous nucleic acids
encoding a pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase,
a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H
2 hydrogenase; or wherein said microbial organism comprising (iii) comprises two exogenous
nucleic acids encoding a CO dehydrogenase and an H
2 hydrogenase.
[0177] In some aspects, the non-naturally occurring microbial organism as disclosed herein
includes, wherein said at least one exogenous nucleic acid is a heterologous nucleic
acid. In some aspects, the non-naturally occurring microbial organism is in a substantially
anaerobic culture medium.
[0178] In some embodiments, the invention provides a method for producing 1,3-butadiene,
comprising culturing a non-naturally occurring microbial organism as disclosed herein
under conditions and for a sufficient period of time to produce 1,3-butadiene.
[0179] In some embodiments, the invention provides a non-naturally occurring microbial organism,
comprising a microbial organism haying a 2,4-pentadienoate pathway comprising at least
one exogenous nucleic acid encoding a 2,4-pentadienoate pathway enzyme expressed in
a sufficient amount to produce 2,4-pentadienoate, said 2,4-pentadienoate pathway selected
from: (A) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase
(aldehyde forming); a 3,5-dioxopentanoate reductase (aldehyde reducing); a 5-hydroxy-3-oxopentanoate
reductase; a 3,5-dihydroxypentanoate dehydratase; and a 5-hydroxypent-2-enoate dehydratase;
(B) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (CoA.
reducing and alcohol forming); a 5-hydroxy-3-oxopentanoate reductase; a 3,5-dihydroxypentanoate
dehydratase; and a 5-hydroxypent-2-enoate dehydratase; (C) a malonyl-CoA:acetyl-CoA
acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde forming); a 3,5-dioxopentanoate
reductase (ketone reducing); a 3-hydroxy-5-oxopentanoate reductase; a 3,5-dihydroxypentanoate
dehydratase; and a 5-hydroxypent-2-enoate dehydratase; (D) a malonyl-CoA:acetyl-CoA
acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing); a 3-hydroxyglutaryl-CoA
reductase (aldehyde forming); a 3-hydroxy-5-oxopentanoate reductase; a 3,5-dihydroxypentanoate
dehydratase; and a 5-hydroxypent-2-enoate dehydratase; and (E) a malonyl-CoA:acetyl-CoA
acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing); a 3-hydroxyglutaryl-CoA
reductase (alcohol forming); a 3,5-dihydroxypentanoate dehydratase; and a 5-hydroxypent-2-enoate
dehydratase.
[0180] In some aspects, the non-naturally occurring microbial organism as disclosed above
comprises two, three, four, five or six exogenous nucleic acids each encoding a 2,4-pentadienoate
pathway enzyme. For example, the microbial organism comprises exogenous nucleic acids
encoding each of the enzymes selected from: (A) a malonyl-CoA:acetyl-CoA acyltransferase;
a 3-oxoglutaryl-CoA reductase (aldehyde forming); a 3,5-dioxopentanoate reductase
(aldehyde reducing); a 5-hydroxy-3-oxopentanoate reductase; a 3,5-dihydroxypentanoate
dehydratase; and a 5-hydroxypent-2-enoate dehydratase; (B) a malonyl-CoA:acetyl-CoA
acyltransferase; a 3-oxoglutaryl-CoA reductase (CoA reducing and alcohol forming);
a 5-hydroxy-3-oxopentanoate reductase; a 3,5-dihydroxypentanoate dehydratase; and
a 5-hydroxypent-2-enoate dehydratase; (C) a malonyl-CoA:acetyl-CoA acyltransferase;
a 3-oxoglutaryl-CoA reductase (aldehyde forming); a 3,5-dioxopentanoate reductase
(ketone reducing); a 3-hydroxy-5-oxopentanoate reductase; a 3,5-dihydroxypentanoate
dehydratase; and a 5-hydroxypent-2-enoate dehydratase; (D) a malonyl-CoA:acetyl-CoA
acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing); a 3-hydroxyglutaryl-CoA
reductase (aldehyde forming); a 3-hydroxy-5-oxopentanoate reductase; a 3,5-dihydroxypentanoate
dehydratase; and a 5-hydroxypent-2-enoate dehydratase; and (E) a malonyl-CoA:acetyl-CoA
acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing); a 3-hydroxyglutaryl-CoA
reductase (alcohol forming); a 3,5-dihydroxypentanoate dehydratase; and a 5-hydroxypent-2-enoate
dehydratase.
[0181] In some embodiments, the invention provides a non-naturally occurring microbial organism
as disclosed above, wherein said microbial organism further comprises: (i) a reductive
TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA
pathway enzyme, wherein said at least one exogenous nucleic acid is selected from
an ATP-citrate lyase, a citrate lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin
oxidoreductase; (ii) a reductive TCA pathways comprising at least one exogenous nucleic
acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous
nucleic acid is selected from a pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate
carboxylase, a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, an H2 hydrogenase;
or (iii) at least one exogenous nucleic acid encodes an enzyme selected from a CO
dehydrogenase, an H2 hydrogenase, and combinations thereof. In some aspects, the microbial
organism comprising (i) further comprises an exogenous nucleic acid encoding an enzyme
selected from a pyruvate:ferredoxin oxidoreductase, an aconitase, an isocitrate dehydrogenase,
a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase,
an acetate kinase, a phosphotransacetylase, an acetyl-CoA synthetase, an NAD(P)H:ferredoxin
oxidoreductase, ferredoxin, and combinations thereof. In some aspects, the non-naturally
occurring microbial organism comprising (ii) further comprises an exogenous nucleic
acid encoding an enzyme selected from an aconitase, an isocitrate dehydrogenase, a
succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase,
and combinations thereof.
[0182] In some aspects, the microbial organism as disclosed above comprising (i) comprises
four exogenous nucleic acids encoding an ATP-citrate lyase, citrate lyase, a fumarate
reductase, and an alpha-ketoglutarate:ferredoxin oxidoreductase; wherein said microbial
organism comprising (ii) comprises five exogenous nucleic acids encoding a pyruvate:ferredoxin
oxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase,
a CO dehydrogenase, and an H
2 hydrogenase; or wherein said microbial organism comprising (iii) comprises two exogenous
nucleic acids encoding a CO dehydrogenase and an H
2 hydrogenase.
[0183] In some aspects, the non-naturally occurring microbial organism as disclosed herein
includes, wherein said at least one exogenous nucleic acid is a heterologous nucleic
acid. In some aspects, the non-naturally occurring microbial organism is in a substantially
anaerobic culture medium.
[0184] In some embodiments, the invention provides a method for producing 2,4-pentadienoate,
comprising culturing a non-naturally occurring microbial organism as disclosed above
under conditions and for a sufficient period of time to produce 2,4-pentadienoate.
[0185] In some embodiments, the invention provides a non-naturally occurring microbial organism,
comprising a microbial organism having a 3-butene-1-ol pathway comprising at least
one exogenous nucleic acid encoding a 3-butene-1-ol pathway enzyme expressed in a
sufficient amount to produce 3-butene-1-ol, said 3-butene-1-ol pathway selected from:
(A) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde
forming); a 3,5-dioxopentanoate reductase (aldehyde reducing); a 5-hydroxy-3-oxopentanoate
reductase; a 3,5-dihydroxypentanoate dehydratase; and a 5-hydroxypent-2-enoate decarboxylase;
(B) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde
forming); a 3,5-dioxopentanoate reductase (aldehyde reducing); a 5-hydroxy-3-oxopentanoate
reductase; and a 3,5-dihydroxypentanoate decarboxylase; (C) a malony)-CoA:acetyl-CoA
acyltransferase; a 3-oxoglutaryl-CoA reductase (CoA reducing and alcohol forming);
a 5-hydroxy-3-oxopentanoate reductase; a 3,5-dihydroxypentanoate dehydratase; and
a 5-hydroxypent-2-enoate decarboxylase; (D) a matonyl-CoA:acetyl-CoA acyltransferase;
a 3-oxoglutaryl-CoA reductase (CoA reducing and alcohol forming); a 5-hydroxy-3-oxopentanoate
reductase; and a 3,5-dihydroxypentanoate decarboxylase; (E) a malonyl-CoA:acetyl-CoA
acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde forming); a 3,5-dioxopentanoate
reductase (ketone reducing); a 3-hydroxy-5-oxopentanoate reductase; a 3,5-dihydroxypentanoate
dehydratase; and a 5-hydroxypent-2-enoate decarboxylase; (F) a malonyl-CoA:acetyl-CoA
acyltransferase; a 3-oxoglutaryl-CoA-reductase (aldehyde forming); a 3,5-dioxopentanoate
reductase (ketone reducing); a 3-hydroxy-5-oxopentanoate reductase; and a 3,5-dihydroxypentanoate
decarboxylase; (G) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglitaryl-CoA reductase
(ketone-reducing); a 3-hydroxyglutaryl-CoA reductase (aldehyde forming); a 3-hydroxy-5-oxopentanoate
reductase; a 3,5-dihydroxypentanoate dehydratase; and a 5-hydroxypent-2-enoate decarboxylase;
(H) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing);
a 3-hydroxyglutaryl-CoA reductase (aldehyde forming); a 3-hydroxy-5-oxopentanoate
reductase; and a 3,5-dihydroxypentanoate decarboxylase; (I) a malonyl-CoA:acetyl-CoA
acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing); a 3-hydroxyglutaryl-CoA
reductase (alcohol forming); a 3,5-dihydroxypentanoate dehydratase; and a 5-hydroxypent-2-enoate
decarboxylase; and (J) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA
reductase (ketone-reducing); a 3-hydroxyglutaryl-CoA reductase (alcohol forming);
and a 3,5-dihydroxypentanoate decarboxylase.
[0186] In some aspects, the non-naturally occurring microbial organism as dislosed above
comprises two, three, four or five exogenous nucleic acids each encoding a 3-butene-1-ol
pathway enzyme. For example, in some aspects, microbial organism comprises erogenous
nucleic acids encoding each of the enzymes selected from: (A) a malonyl-CoA:acetyl-CoA
acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde forming); a 3,5-dioxopentanoate
reductase (aldehyde reducing); a 5-hydroxy-3-oxopentanoate reductase; a 3,5-dihydroxypentanoate
dehydratase; and a 5-hydroxypent-2-enoate decarboxylase; (B) a malonyl-CoA:acetyl-CoA
acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde forming); a 3,5-dioxopentanoate
reductase (aldehyde reducing); a 5-hydroxy-3-oxopentanoate reductase; and a 3,5-dihydroxypentanoate
decarboxylase; (C) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase
(CoA reducing and alcohol forming); a 5-hydroxy-3-oxopentanoate reductase; a 3,5-dihydroxypentanoate
dehydratase; and a 5-hydroxypent-2-enoate decarboxylase; (D) a malonyl-CoA:acetyl-CoA
acyltransferase; a 3-oxoglutaryl-CoA reductase (CoA reducing and alcohol forming);
a 5-hydroxy-3-oxopentanoate reductase; and a 3,5-dihydroxypentanoate decarboxylase;
(E) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde
forming); a 3,5-dioxopentanoate reductase (ketone reducing); a 3-hydroxy-5-oxopentanoate
reductase; a 3,5-dihydroxypentanoate dehydratase; and a 5-hydroxypent-2-enoate decarboxylase;
(F) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde
forming); a 3,5-dioxopentanoate reductase (ketone reducing); a 3-hydroxy-5-oxopentanoate
reductase; and a 3,5-dihydroxypentanoate decarboxylase; (G) a malonyl-CoA:acetyl-CoA
acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing); a 3-hydroxyglutaryl-CoA
reductase (aldehyde forming); a 3-hydroxy-5-oxopentanoate reductase; a 3,5-dihydroxypentanoate
dehydratase; and a 5-hydroxypent-2-enoate decarboxylase; (H) a malonyl-CoA:acetyl-CoA
acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing); a 3-hydroxyglutaryl-CoA
reductase (aldehyde forming); a 3-hydroxy-5-oxopentanoate reductase; and a 3,5-dihydroxypentanoate
decarboxylase; (I) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase
(ketone-reducing); a 3-hydroxyglutaryl-CoA reductase (alcohol forming); a 3,5-dihydroxypentanoate
dehydratase; and a 5-hydroxypent-2-enoate decarboxylase; and (J) a malonyl-CoA:acetyl-CoA
acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing); a 3-hydroxyglutaryl-CoA
reductase (alcohol forming); and a 3,5-dihydroxypentanoate decarboxylase.
[0187] In some embodiments, the invention provides a non-naturally occurring microbial organism
as disclosed above, wherein said microbial organism further comprises: (i) a reductive
TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA
pathway enzyme, wherein said at least one exogenous nucleic acid is selected from
an ATP-citrate lyase, a citrate lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin
oxidoreductase; (ii) a reductive TCA pathway comprising at least one exogenous nucleic
acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous
nucleic acid is selected from a pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate
carboxylase, a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H2 hydrogenase;
or (iii) at least one exogenous nucleic acid encodes an enzyme selected from a CO
dehydrogenase, an H2 hydrogenase, and combinations thereof. In some aspects, the non-naturally
occurring microbial organism comprising (i) further comprises an exogenous nucleic
acid encoding an enzyme selected from a pyruvate:ferredoxin oxidoreductase, an aconitase,
an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase,
a fumarase, a malate dehydrogenase, an acetate kinase, a phosphotransacetylase, an
acetyl-CoA synthetase, an NAD(P)H:ferredoxin oxidoreductase, ferredoxin, and combinations
thereof. In some aspects, the non-naturally occurring microbial organism comprising
(ii) further comprises an exogenous nucleic acid encoding an enzyme selected from
an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA
transferase, a fumarase, a malate dehydrogenase, and combinations thereof. In some
aspects, the non-naturally occurring microbial comprising (i) comprises four exogenous
nucleic acids encoding an ATP-citrate lyase, citrate lyase, a fumarate reductase,
and an alpha-ketoglutarate:ferredoxin oxidoreductase; wherein said microbial organism
comprising (ii) comprises five exogenous nucleic acids encoding a pyruvate:ferredoxin
oxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase,
a GO dehydrogenase, and an H
2 hydrogenase; or wherein said microbial organism comprising (iii) comprises two exogenous
nucleic acids encoding a CO dehydrogenase and an H
2 hydrogenase.
[0188] In some aspects, the non-naturally occurring microbial organism as disclosed herein
includes, wherein said at least one exogenous nucleic acid is a heterologous nucleic
acid. In some aspects, the non-naturally occurring microbial organism is in a substantially
anaerobic culture medium.
[0189] In some embodiments, the invention provides a method for producing 3-butene-1-ol,
comprising culturing a non-naturally occurring microbial organism as disclosed above
under conditions and for a sufficient period of time to produce 3-butene-1-ol.
[0190] In some embodiments, the invention provides a method for producing 1,3-butadiene
comprising, culturing a non-naturally occurring microbial organism that can produce
3-butene-1-ol as disclosed above under conditions and for a sufficient period of time
to produce 3-butene-1-ol, and chemically converting said 3-butene-1-ol to 1,3-butadiene.
It is understood that methods for chemically converting 3-butene-1-ol to 1,3-butadiene
are well know in the art.
[0191] This invention is also directed, in part to engineered biosynthetic pathways to improve
carbon flux through a central metabolism intermediate en route to 2,4-pentadienoate,
3-butene-1-ol, or 1,3-butadiene. The present invention provides non-naturally occurring
microbial organisms having one or more exogenous genes encoding enzymes that can catalyze
various enzymatic transformations en route to 2,4-pentadienoate, 3-butene-1-ol, or
1,3-butadiene. In some embodiments, these enzymatic transformations are part of the
reductive tricarboxylic acid (RTCA) cycle and are used to improve product yields,
including but not limited to, from carbohydrate-based carbon feedstock.
[0192] In numerous engineered pathways, realization of maximum product yields based on carbohydrate
feedstock is hampered by insufficient reducing equivalents or by loss of reducing
equivalents and/or carbon to byproducts. In accordance with some embodiments, the
present invention increases the yields of 2,4-pentadienoate, 3-butene-1-ol, or 1,3-butadiene
by (i) enhancing carbon fixation via the reductive TCA cycle, and/or (ii) accessing
additional reducing equivalents from gaseous carbon sources and/or syngas components
such as CO, CO
2, and/or H
2. In addition to syngas, other sources of such gases include, but are not limted to,
the atmosphere, either as found in nature or generated.
[0193] The CO
2-fixing reductive tricarboxylic acid (RTCA) cycle is an endergenic anabolic pathway
of CO
2 assimilation which uses reducing equivalents and ATP (Figure 22). One turn of the
RTCA cycle assimilates two moles of CO
2 into one mole of acetyl-CoA, or four moles of CO
2 into one mole of oxaloacetate. This additional availability of acetyl-COA improves
the maximum theoretical yield of product molecules derived from carbohydrate-based
carbon feedstock. Exemplary carbohydrates include but are not limited to glucose,
sucrose, xylose, arabinose and glycerol.
[0194] In some embodiments, the reductive TCA cycle, coupled with carbon monoxide dehydrogenase
and/or hydrogenase enzymes, can be employed to allow syngas, CO
2, CO, H
2, and/or other gaseous carbon, source utilization by microorganisms. Synthesis gas
(syngas), in particular is a mixture of primarily H
2 CO, sometimes including some amounts of CO
2, that can be obtained via gasification of any organic feedstock, such as coal, coal
oil, natural gas, biomass, or waste organic matter. Numerous gasification processes
have been developed, and most designs are based on partial oxidation, where limiting
oxygen avoids full combustion, of organic materials at high temperatures (500-1500°C)
to provide syngas as a 0.5:1-3:1 H
2/CO mixture. In addition to coal, biomass of many types has been used for syngas production
and represents an inexpensive and flexible feedstock for the biological production
of renewable chemicals and fuels. Carbon dioxide can be provided from the atmosphere
or in condensed from, for example, from a tank cylinder, or via sublimation of solid
CO
2. Similarly, CO and hydrogen gas can be provided in reagent farm and/or mixed in any
desired ratio. Other gaseous carbon forms can include, for example, methanol or Similar
volatile organic solvents.
[0195] The components of synthesis gas and/or other carbon sources can provide sufficient
CO
2, reducing equivalents, and ATP for the reductive TCA cycle to operate. One turn of
the RTCA cycle assimilates two moles of CO
2 into one mole of acetyl-CoA and requires 2 ATP and 4 reducing equivalents. CO and/or
H
2 can provide reducing equivalents by means of carbon monoxide dehydrogenase and hydrogenase
enzymes, respectively. Reducing equivalents can come in the form of NADH, NADPH, FADH,
reduced quinones, reduced ferredoxins, reduced flavodoxins and thioredoxins. The reducing
equivalents, particularly NADH, NADPH, and reduced ferredoxin, can serve as cofactors
for the RTCA cycle enzymes, for example, malate dehydrogenase, fumarate reductase,
alpha-ketoglutarate:ferredoxin oxidoreductase (alternatively known as 2-oxoglutarate:ferredoxin
oxidoreductase, alpha-ketoglutarate synthase, or 2-oxoglutarate synthase), pyravate:fettedoxin
oxidoreductase and isocitrate dehydrogenase. The electrons from these reducing equivalents
can alternatively pass through an ion-gradient producing electron transport chain,
where they are passed to an acceptor such as oxygen, nitrate, oxidized metal ions,
protons, or an electrode. The ion-gradient can then be used for ATP generation via
an ATP synthase or similar enzyme.
[0197] The key carbon-fixing enzymes of the seductive TCA cycle are alpha-ketoglutarate:ferredoxin
oxidoreductase, pyruvate:ferredoxin oxidoreductase and isocitrate dehydrogenase. Additional
carbon may be fixed during the conversion of phosphoenolpyruvate to oxaloacetate by
phosphoenolpyruvate carboxylase or phosphoenolpyruvate carboxykinase carboxykinase
or by conversion of pyruvate to malate by malic enzyme.
[0198] Many of the enzymes in the TCA cycle are reversible and can catalyze reactions in
the reductive and oxidative directions. However, some TCA cycle reactions are irreversible
in vivo and thus different enzymes are used to catalyze these reactions in the directions
required for the reverse TCA cycle. These reactions are: (1) conversion of citrate
to oxaloacetate and acetyl-CoA, (2) conversion of fumarate to succinate, and (3) conversion
of succinyl-CoA to alpha-ketoglutarale. In the TCA cycle, citrate is formed from the
condensation of oxaloacetate and acetyl-CoA. The reverse reaction, cleavage of citrate
to oxaloacetate and acetyl-CoA, is ATP-dependent and catalyzed by ATP-citrate lyase,
or citryl-CoA synthetase and citryl-CoA lyase. Alternatively, citrate lyase can be
coupled to acetyl-CoA synthetase, an acetyl-CoA transferase, or phosphotransacetylase
and acetate kinase to form acetyl-CoA and oxaloacetate from citrate. The conversion
of succinate to fumarate is catalyzed by succinate dehydrogenase while the reverse
reaction is catalyzed by fumarate reductase. In the TCA cycle succinyl-CoA is formed
from the NAD(P)
+ dependent decarboxylation of alpha-ketoglutarate by the alpha-ketoglutarate dehydrogenase
complex. The reverse reaction is catalyzed by alpha-ketoglutarate:ferredoxin oxidoreductase.
[0199] An organism capable of utilizing the reverse tricarboxylic acid cycle to enable production
of acetyl-CoA-derived products on 1) CO, 2) CO
2 and H
2, 3) GO and CO
2, 4) synthesis gas comprising CO and H
2, and 5) synthesis gas or other gaseous carbon sources comprising CO, CO
2, and H
2 can include any of the following enzyme activities: ATP-citrate lyase, citrate lyase,
aconitase, isocitrate dehydrogenase, alpha-ketoglutarate:ferredoxin oxidoreductase,
succinyl-CoA synthetase, succinyl-CoA transfera, fumarate reductase, fumarase, malate
dehydrogenase, acetate kinase, phosphotransacetylase, acetyl-CoA synthetase, acetyl-CoA
transferase, pyruvate:ferredoxin oxidoreductase, NAD(P)H:ferredoxin oxidoreductase,
carbon monoxide dehydrogenase, hydrogenase, and ferredoxin (see Figure 23). Enzymes
and the corresponding genes required for these activities are described herein above.
[0200] Carbon from syngas or other gaseous carbon sources can be fixed via the reverse TCA
cycle and components thereof. Specifically, the combination of certain carbon gas-utilization
pathway components with the pathways for formation of 2,4-pentadienoate, 3-butene-1-ol,
or 1,3-butadiene from acetyl-CoA results in high yields of these products by providing
an efficient mechanism for fixing the carbon present in carbon dioxide, fed exogenously
or produced endogenously from CO, into acetyl-CoA.
[0201] In some embodiments, a 2,4-pentadienoate, 3-butene-1-ol, or 1,3-bittadiene pathway
in a non-natlirally occurring microbial organism of the invention can utilize any
combination of (1) CO, (2) CO
2, (3) H
2, or mixtures thereof to enhance the yields of biosynthetic steps involving reduction,
including addition to driving the reductive TCA cycle.
[0202] In some embodiments a non-naturally occurring microbial organism having an 2,4-pentadienoate,
3-butene-1-ol, or 1,3-butadiene pathways includes at least one exogenous nucleic acid
encoding a reductive TCA pathway enzyme. The at least one exogenous nucleic acid is
selected from an ATP-citrate lyase, citrate lyase, a fumarate reductase, isocitrate
dehydrogenase, aconitase, and an alpha-ketoglutarate:ferredoxin oxidoreductase; and
at least one exogenous enzyme selected from a carbon monoxide dehydrogenase, a hydrogenase,
a NAD(P)H:ferredoxin oxidoreductase, and a ferredoxin, expressed in a sufficient amount
to allow the utilization of (1) CO, (2) CO
2, (3) H
2, (4) CO
2 and H
2, (5) CO and CO
2, (6) CO and H
2, or (7) CO, CO
2, and H
2.
[0203] In some embodiments a method includes culturing a non-naturally occurring microbial
organism having a 2,4-pentadienoate, 3-butene-1-ol, or 1,3-butadiene pathway also
comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme.
The at least one exogenous nucleic acid is selected from an ATP-citrate lyase, citrate
lyase, a fumarate reductase, isocitrate dehydrogenase, aconitase, and an alpha-ketoglutarate:ferredoxin
oxidoreductase. Additionally, such an organism can also include at least one exogenous
enzyme selected from a carbon monoxide dehydrogenase, a hydrogenase, a NAD(P)H:ferredoxin
oxidoreductase, and a ferredoxin, expressed in a sufficient amount to allow the utilization
of (1) CO, (2) CO
2, (3) H
2, (4) CO
2 and H
2, (5) CO and CO
2, (6) CO and H
2, or (7) CO, CO
2, and H
2 to produce a product.
[0204] In some embodiments a non-naturally occurring microbial organism having an 2,4-pentadienoate,
3-butene-1-ol, or 1,3-butadiene pathway further includes at least one exogenous nucleic
acid encoding a reductive TCA pathway enzyme expressed in a sufficient amount to enhance
carbon flux through acetyl-CoA. The at least one exogenous nucleic acid is selected
from an ATP-citrate lyase, citrate lyase, a fumarate reductase, a pyruvate:ferredoxin
oxidoreductase, isocitrate dehydrogenase, aconitase and an alpha-ketoglutarate:ferredoxin.
oxidoreductase.
[0205] In some embodiments a non-naturally occurring microbial organism having an 2,4-pentadienoate,
3-butene-1-ol, or 1,3-butadiene pathway includes at least one exogenous nucleic acid
encoding an enzyme expressed in a sufficient amount to enhance the availability of
reducing equivalents in the presence of carbon monoxide and/or hydrogen, thereby increasing
the yield of redox-limited products via carbohydrate-based carbon feedstock. The at
least one exogenous nucleic acid is selected from a carbon monoxide dehydrogenase,
at hydrogenase, an NAD(P)H:ferredoxin oxidoreductase, and a ferredoxin. In some embodiments,
the present invention provides a method for enhancing the availability of reducing
equivalents in the presence of carbon monoxide or hydrogen thereby increasing the
yield of redox-limited products via carbohydrate-based carbon feedstock, such as sugars
or gaseous carbon sources, the method includes culturing this non-naturally occurring
microbial organism under conditions and for a sufficient period of time to produce
2,4-pentadienoate, 3-butene-1-ol, or 1,3-butadiene.
[0206] In some embodiments, the non-naturally occurring microbial organism having an 2,4-pentadienoate,
3-butene-1-ol, or 1,3-butadiene pathway includes two exogenous nucleic acids, each
encoding a reductive TCA pathway enzyme. In some embodiments, the non-naturally occurring
microbial organism having an 2,4-pentadienoate, 3-butene-1-ol, or 1,3-butadiene pathway
includes three exogenous nucleic acids each encoding a reductive TCA pathway enzyme.
In some embodiments, the non-naturally occurring microbial organism includes three
exogenous nucleic acids encoding an ATP-citrate lyase, a fumarate reductase, and an
alpha-ketoglutarate:ferredoxin oxidoreductase. In some embodiments, the non-naturally
occurring microbial organism includes three exogenous nucleic acids encoding a citrate
lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin oxidoreductase.
[0207] In some embodiments, the non-naturally occurring microbial organisms having an 2,4-pentadienoate,
3-butene-1-ol, or 1,3-butadiene pathway further include an exogenous nucleic acid
encoding an enzyme selected from a pyruvate:ferredoxin oxidoreductase, an aconitase,
an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase,
a fumarase, a malate dehydrogenase, an acetate kinase a phosphotransacetylase, an
acetyl-CoA synthetase, an NAD(P)H:ferredoxin oxidoreductase, and combinations thereof.
[0208] In some embodiments, the non-naturally occurring microbial organism having an 2,4-pentadienoate,
3-butene-1-ol, or 1,3-butadiene pathway further includes an exogenous nucleic pentadienoate,
3-butene-1-ol, or 1,3-butadiene pathway further includes an exogenous nucleic acid
encoding an enzyme selected from carbon monoxide dehydrogenase, acetyl-CoA synthase,
ferredoxin, NAD(P)H:ferredoxin oxidoreductase and combinations thereof.
[0209] In embodiments, the non-naturally occurring microbial organism having an 2,4-pentadienoate,
3-butene-1-ol, or 1,3-butadiene pathway utilizes a carbon feedstock selected from
(1) CO, (2) CO
2, (3) CO
2 and H
2, (4) CO and H
2, or (5) CO, CO
2, and H
2. In some embodiments, the non-naturally occurring microbial organism having an 2,4-pentadienoate,
3-butene-1-ol, or 1,3-butadiene pathway utilizes hydrogen for reducing equivalents.
In some embodiments, the non-naturally occurring microbial organism having an 2,4-pentadienoate,
3-butene-1-ol, or 1,3-butadiene pathway utilizes CO for reducing equivalents. In some
embodiments, the non-naturally occurring microbial organism having an 2,4-pentadienoate,
3-butene-1-ot, or 1,3-butadiene pathway utilizes combinations of CO and hydrogen for
reducing equivalents.
[0210] In some embodiments, the non-naturally occurring microbial organism having an 2,4-pentadienoate,
3-butene-1-ol, or 1,3-butadiene pathway further includes one or more nucleic acids
encoding an enzyme selected from a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate
carboxykinase, a pyruvate carboxylase, and a malic enzyme.
[0211] In some embodiments, the non-naturally occurring microbial organism having an 2,4-pentadienoate,
3-butene-1-ol, or 1,3-butadiene pathway further includes one or more nucleic acids
encoding an enzyme selected from a malate dehydrogenase, a fumarase, a fumarate reductase,
a succinyl-CoA synthetase, and a succinyl-CoA transferase.
[0212] In some embodiments, the non-naturally occurring microbial organism having an 2,4-pentadienoate,
3-butene-1-ol, or 1,3-butadiene pathway further includes at least one exogenous nucleic
acid encoding a citrate lyase, an ATP-citrate lyase, a citryl-CoA synthetase, a citryl-CoA
lyase an aconitase, an isocitrate dehydrogenase, a succinyl-CoA. synthetase, a succinyl-CoA
transferase, a fumarase, a malate dehydrogenase, an acetate kinase, a phosphotransacetylase,
an acetyl-CoA synthetase, and a ferredoxin.
[0213] In some embodiments, the carbon feedstock and other cellular uptake sources such
as phosphate, ammonia, sulfate, chloride and other halogens can be chosen to alter
the isotopic distribution of the atoms present in 1,3-butadiene or any 1,3-butadiene
pathway intermediate. The various carbon feedstock and other uptake sources enumerated
above will be referred to herein, collectively, as "uptake sources." Uptake sources
can provide isotopic enrichment for any atom present in the product toluene, benzene,
p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate,
benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol or 1,3-butadiene pathway or any
intermediate en route thereto. The various carbon feedstock and other uptake sources
enumerated above will be referred to herein, collectively, as "uptake sources." Uptake
sources can provide isotopic enrichment for any atom present in the product toluene,
benzene, p-totuate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate,
benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol or 1,3-butadiene or toluene, benzene,
p-toluate, terephthatate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate,
benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol or 1,3-butadiene pathway intermediate
including any toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate,
(2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol
or 1,3-butadiene impurities generated in diverging away from the pathways at any point.
Isotopic enrichment can be achieved for any target atom including, for example, carbon,
hydrogen, oxygen, nitrogen, sulfur, phosphorus, chloride or other halogens.
[0214] In some embodiments, the uptake sources can be selected to alter the carbon-12, carbon-13,
and carbon-14 ratios. In some embodiments, the uptake sources can be selected to alter
the oxygen-16, oxygen-17, and oxygen-18 ratios. In some embodiments, the uptake sources
can be selected to alter the hydrogen, deuterium, and tritium ratios. In some embodiments,
the uptake sources can selected to alter the nitrogen-14 and nitrogen-15 ratios. In
some embodiments, the uptake sources can be selected to alter the sulfur-32, sulfur-33,
sulfur-34, and sulfur-35 ratios. In some embodiments, the uptake sources can be selected
to alter the phosphorus-31, phosphorus-32, and phosphorus-33 ratios. In some embodiments,
the uptake sources can be selected to alter the chlorine-35, chlorine-36, and chlorine-37
ratios.
[0215] In some embodiments, a target isotopic ratio of an uptake source can be obtained
via synthetic chemical enrichment of the uptake source. Such isotopically enriched
uptake sources can be purchased commercially or prepared in the laboratory. In some
embodiment, a target isotopic ratio of an uptake source can be obtained by choice
of origin of the uptake source in nature, In some such embodiments, a source of carbon,
for example, can be selected from a fossil fuel-derived carbon source, which can be
relativity depleted of carbon-14, or an environmental carbon source, such as CO
2, which can possess a larger amount of carbon-14 than its petroleum-derived counterpart.
[0216] Isotopic enrichment is readily assessed by mass spectrometry using techniques known
in the art such as Stable Isotope Ratio Mass Spectrometry (SIRMS) and Site-Specific
Natural Isotopic Fractionation by Nuclear Magnetic Resonance (SNIF-NMR). Such mass
spectral techniques can be integrated with separation techniques such as liquid chromatography
(LC) and/or high performance liquid chromatography (HPLC).
[0217] In some embodiments, the present invention provides toluene, benzene, p-toluate,
terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate,
benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol or 1,3-butadiene or a toluene,
benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate,
benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol or 1,3-butadiene intermediate that
has a carbon-12, carbon-13, and carbon-14 ratio that reflects an atmospheric carbon
uptake source. In some such embodiments, the uptake source is CO
2. In some embodiments, In some embodiments, the present invention provides toluene,
benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate,
benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol or 1,3-butadiene, intermediate
that has a carbon-12, carbon-13, and carbon-14 ratio that reflects petroleum-based
carbon uptake source. In some embodiments, the present invention provides toluene,
benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate,
benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol or 1,3-butadiene or a toluene,
benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate,
benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol or 1,3-butadiene intermediate,
that has a carbon-12, carbon-13, and carbon-14 ratio that is obtained by a combination
of an atmospheric carbon uptake source with a petroleum-based uptake source. Such
combination of uptake sources is one means by which the carbon-12, carbon-13, and
carbon-14 ratio can be varied.
[0218] Accordingly, given the teachings and guidance provided herein, those skilled in the
art will understand, that a non-naturally occurring microbial organism can be produced
that secretes the biosynthesized compounds of the invention when grown on a carbon
source such as a carbohydrate. Such compounds include, for example, toluene, benzene
p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate,
benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol or 1,3-butadiene and any of the
intermediate metabolites in the toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate,
(2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol
or 1,3-butadiene pathway. All that is required is to engineer in one or more of the
required enzyme or protein activities to achieve biosynthesis of the desired compound
or intermediate including, for example, inclusion of some or all of the toluene, benzene,
p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate,
benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol or 1,3-butadiene biosynthetic pathways.
Accordingly, the invention provides a non-naturally occurring microbial organism that
produces and/or secretes toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate,
(2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol
or 1,3-butadiene when grown on a carbohydrate or other carbon source and produces
and/or secretes any of the intermediate metabolites shown in the toluene, benzene,
p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate,
benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol or 1,3-butadiene pathway when grown
on a carbohydrate or other carbon source. The toluene, benzene, p-toluate, terephthalate,
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate,
styrene, 2,4-pentadienoate, 3-butene-1ol or 1,3-butadiene producing microbial organisms
of the invention can initiate synthesis from an intermediate, for example, phenylalanine,
phenylpyruvate, phenylacetaldehyde, phenylacetate, benzoyl-CoA, 3-oxo-3-phenylpropionyl-CoA,
[(3-oxo-3-phenylpropionyl)oxy] phosphonate, benzoyl acetate, acetophenone, 1-phenylethanol,
trans,trans-muconate,
cis,
trans-muconate,
cis,cis-muconate,
trans-2,4-pentadienoate, and
cis,-2,4-pentadienoate. As a further example, a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate
pathway intermediate can be 1-deoxy-D-xylulose-5-phosphate or C-methyl-D-erythritol-4-pbosphate
(see Example III and Figure 5). A p-toluate pathway intermediate can be, for example,
2,4-dihydroxy-5-methyl-6-[(phosphonooxy)methyl]oxane-2-carboxylate, 1,3-dihydroxy-4-methyl-5-oxocyclohexane-1-carboxylate,
5-hydroxy-4-methyl-3-oxocyclohex-1-ene-1-carboxylate, 3,5-dihydroxy-4-methylcyclohex-1-ene-1-carboxylate,
5-hydroxy-4-methyl-3-(phosphonooxy)cyclohex-1-ene-1-carboxylate, 5-[(1-carboxyeth-1-en-1-yl)oxy]-4-methyl-3-(phospbonooxy)cyclohex-1-ene-1-carboxylate,
or 3-[(1-carboxyeth-1-en-1-yl)oxy]-4-methylcyclohexa-1,5-diene-1-carboxylate (see
Example IV and Figure 6). A terephthatate intermediate can be, for example, 4-carboxybenzyl
alcohol or 4-carboxybenzaldehyde (see Example V and Figure 7).
[0219] As disclosed herein, p-toluate and benzoate are exemplary intermediates that can
be the subject of a non-naturally occurring microbial organism. Such carboxylates
can occur in ionized form or fully protonated form. Accordingly, the suffix "-ate,"
or the acid form, can be used interchangeably to describe both the free acid form
as well as any deprotonated form, in particular since the ionized form is known to
depend on the pH in which the compound is found. It is understood that propionate
products accessible in accordance with the present invention include ester forms,
such as O-carboxylate and S-carboxylate esters. O- and S-carboxylates can include
lower alkyl, that is C1 to C6, branched or straight chain carboxylates. Some such
O- or S-carboxylates include, without limitation, methyl, ethyl, n-propyl, n-butyl,
i-propyl, sec-butyl, and tert-butyl, pentyl, hexyl O- or S-carboxylates, any of which
can further possess an unsaturation, providing for example, propenyl, butenyl, pentyl,
and hexenyl O- or S-carboxylates. O-carboxylates can be the product of a biosynthetic
pathway. Exemplary O-carboxylates accessed via biosynthetic pathways can include,
without limitation, methyl propionate, ethyl propionate, and n-propyl propionate.
Other biosynthetically accessible O-propionates can include medium to long chain groups,
that is C7-C22, O-propionate esters derived from fatty alcohols, such heptyl, octyl,
nonyl, decyl, undecyl, lauryl, tridecyl, myristyl, pentadecyl, cetyl, palmitolyl,
heptadecyl, stearyl, nonadecyl, arachidyl, heneicosyl, and behenyl alcohols, any one
of which can be optionally branched and/or contain unsaturations. O-propionate esters
can also be accessed via a biochemical or chemical process, such as esterification
of a free carboxylic acid product or transesterification of an O- or S-propionate.
S-carboxylates are exemplified by CoA S-esters, cysteinyl S-esters, alkylthioesters,
and various aryl and heteroaryl thioesters.
[0220] The non-naturally occurring microbial organisms of the invention are constructed
using methods well known in the art as exemplified herein to exogenously express at
least one nucleic acid encoding a toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate,
(2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol
or 1,3-butadiene pathway enzyme or protein in sufficient amounts to produce toluene,
benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate,
benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene . It is understood
that the microbial organisms of the invention are cultured under conditions sufficient
to produce toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate,
(2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol
or 1,3-butadiene Following the teachings and guidance provided herein, the non-naturally
occurring microbial organisms of the invention can achieve biosynthesis of toluene,
benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate,
benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene resulting in intracellular
concentrations between about 0.1-200 mM or more. Generally, the intracellular concentration
of toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate,
(2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol
or 1,3-butadiene is between about 3-150 mM, particularly between about 5-125 mM and
more particularly between about 8-100 mM, including about 10 mM, 20 mM, 50 mM, 80
mM, or more. Intracellular concentrations between and above each of these exemplary
ranges also can be achieved from the non-naturally occurring microbial organisms of
the invention.
[0221] In some embodiments, culture conditions include anaerobic or substantially anaerobic
growth or maintenance conditions. Exemplary anaerobic conditions have been described
previously and are well known in the art. Exemplary anaerobic conditions for fermentation
processes are described herein and are described, for example, in
U.S. publication 2009/0047719, filed August 10, 2007. Any of these conditions can be employed with the non-naturally occurring microbial
organisms as well as other anaerobic conditions well known in the art. Under such
anaerobic or substantially anaerobic conditions, the toluene, benzene, p-toluate,
terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate,
benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene producers can
synthesize toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate,
(2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol
or 1,3-butadiene at intracellular concentrations of 5-10 mM or more as well as all
other concentrations exemplified herein. It is understood that, even though the above
description refers to intracellular concentrations, toluene, benzene, p-toluate, terephthalate,
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate,
styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene producing microbial organisms
can produce toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate,
(2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol
or 1,3-butadiene intracellularly and/or secrete the product into the culture medium.
[0222] In addition to the culturing and fermentation conditions disclosed herein, growth
condition for achieving biosynthesis of toluene, benzene, p-toluate, terephthalate,
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate,
styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene can include the addition
of an osmoprotectant to the culturing conditions. In certain embodiments, the non-naturally
occurring microbial organisms of the invention can be sustained, cultured or fermented
as described herein in the presence of an osmoprotectant. Briefly, an osmoprotectant
refers to a compound that acts as an osmolyte and helps a microbial organism as described
herein survive osmotic stress. Osmoprotectants include, but are not limited to, betaines,
amino acids, and the sugar trehalose. Non-limiting examples of such are glycine betaine,
praline betaine, dimethylthetin, dimethylslfonioproprionate, 3-dimethylsulfonio-2-methylproprionate,
pipecolic acid, dimethylsulfonioacetate, choline, L-carnitine and ectoine. In one
aspect, the osmoprotectant is glycine betaine. It is understood to one of ordinary
skill in the art that the amount and type of osmoprotectant suitable for protecting
a microbial organism described herein from osmotic stress will depend on the microbial
organism used. The amount of osmoprotectant in the culturing conditions can be, for
example, no more than about 0.1 mM, no more than about 0.5 mM, no more than about
1.0 mM, no more than about 1.5 mM, no more than about 2.0 mM, no more than about 2.5
mM, no more than about 3.0 mM, no more than about 5.0 mM, no more than about 7.0 mM,
no more than about 10mM, no more than about 50mM, no more than about 100mM or no more
than about 500mM.
[0223] The culture conditions can include, for example, liquid culture procedures as well
as fermentation and other large scale culture procedures. As described herein, particularly
useful yields of the biosynthetic products of the invention can be obtained under
anaerobic or substantially anaerobic culture conditions.
[0224] As described herein, one exemplary growth condition for achieving biosynthesis of
toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate,
(2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol
or 1,3-butadiene includes anaerobic culture or fermentation conditions. In certain
embodiments, the non-naturally occurring microbial organisms of the invention can
be sustained, cultured or fermented under anaerobic or substantially anaerobic conditions.
Briefly, anaerobic conditions refers to an environment devoid of oxygen. Substantially
anaerobic conditions include, for example, a culture, batch fermentation or continuous
fermentation such that the dissolved oxygen concentration in the medium remains between
0 and 10% of saturation. Substantially anaerobic conditions also includes growing
or rusting cells in liquid medium or on solid agar inside a sealed chamber maintained
with an atmosphere of less than 1% oxygen. The percent of oxygen can be maintained
by, for example, sparging the culture with an N
2/CO
2 mixture or other suitable non-oxygen gas or gases.
[0225] The culture conditions described herein can be scaled up and grown continuously for
manufacturing of toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate,
(2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol
or 1,3-butadiene . Exemplary growth procedures include, for example, fed-batch fermentation
and batch separation; fed-batch fermentation and continuous separation, or continuous
fermentation and continuous separation. All of these processes are well known in the
art. Fermentation procedures are particularly useful for the biosynthetic production
of commercial quantities of toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate,
(2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol
or 1,3-butadiene . Generally, and as with non-continuous culture procedures, the continuous
and/or near-continuous production of toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate,
(2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol
or 1,3-butadiene will include culturing a non-naturally occurring toluene, benzene,
p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate,
benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene producing organism
of the invention in sufficient nutrients and medium to sustain and/or nearly sustain
growth in an exponential phase. Continuous culture under such conditions can be include,
for example, growth for 1 day, 2, 3, 4, 5, 6 or 7 days or more. Additionally, continuous
culture can include longer time periods of 1 week, 2, 3, 4 or 5 or more weeks and
up to several months. Alternatively, organisms of the invention can be cultured for
hours, if suitable for a particular application. It is to be understood that the continuous
and/or near-continuous culture conditions also can include all time intervals in between
these exemplary periods. It is further understood that the time of culturing the microbial
organism of the invention is for a sufficient period of time to produce a sufficient
amount of product for a desired purpose.
[0226] Fermentation procedures are well known in the art. Briefly, fermentation for the
biosynthetic production of toluene, benzene, p-toluate, terephthatate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate,
(2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol
or 1,3-butadiene can be utilized in, for example, fed-batch fermentation and batch
separation; fed-batch fermentation and continuous separation, or continuous fermentation
and continuous separation. Examples of batch and continuous fermentation procedures
are well known in the art.
[0227] In addition to the above fermentation procedures using the toluene, benzene, p-toluate,
terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate,
benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene producers of the
invention for continuous production of substantial quantities of toluene, benzene,
p-toluate, terephthatate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate,
benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol or 1,3-butadiene , the toluene,
benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate,
benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol or 1,3-butadiene producers also
can be, for example, simultaneously subjected to chemical synthesis procedures to
convert the product to other compounds or the product can be separated from the fermentation
culture and sequentially subjected to chemical conversion to convert the product to
other compounds, if desired.
[0228] To generate better producers, metabolic modeling can be utilized to optimize growth
conditions. Modeling can also be used to design gene knockouts that additionally optimize
utilization of the pathway (see, for example, U.S. patent publications
US 2002/0012939,
US 2003/0224363,
US 2004/0029149,
US 2004/0072723,
US 2003/0059792,
US 2002/0168654 and
US 2004/0009466, and
U.S. Patent No. 7,127,379). Modeling analysis allows reliable predictions of the effects on cell growth of
shifting the metabolism towards more efficient production of toluene, benzene, p-toluate,
terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate,
benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol or 1,3-butadiene.
[0229] One computational method for identifying and designing metabolic alterations favoring
biosynthesis of a desired product is the OptKnock computational framework (
Burgard et al., Biotechnol. Bioeng. 84:647-657 (2003)). OptKnock is a metabolic modeling and simulation program that suggests gene deletion
or disruption strategies that result in genetically stable microorganisms which overproduce
the target product. Specifically, the framework examines the complete metabolic and/or
biochemical network of a microorganism. in order to suggest genetic manipulations
that force the desired biochemical to become an obligatory byproduct of cell growth.
By coupling biochemical production with cell growth through strategically placed gene
deletions or other functional gene disruption, the growth selection pressures imposed
on the engineered strains after long periods of time in a bioreactor lead to improvement
in performance as a result of the compulsory growth-coupled biochemical production.
Lastly, when gene deletions are constructed there is a negligible possibility of the
designed strains reverting to their wild-type states because the genes selected by
OptKnock are to be completely removed from the genome. Therefore, this computational,
methodology can be used to either identify alternative pathways that lead to biosynthesis
of a desired product or used in connection with the non-naturally occurring microbial
organisms for further optimization of biosynthesis of a desired product.
[0230] Briefly, OptKnock is a term used herein to refer to a computational method and system
for modeling cellular metabolism. The OptKnock program relates to a framework of models
and methods that incorporate particular constraints into flux balance analysis (FBA)
models. These constraints include, for example, qualitative kinetic information, qualitative
regulatory information, and/or DNA microarray experimental data. OptKnock also computes
solutions to various metabolic problems by, for example, tightening the flux boundaries
derived through flux balance models and subsequently probing the performance limits
of metabolic networks in the presence of gene additions or deletions. OptKnock computational
framework allows the construction of model formulations that allow an effective query
of the performance limits of metabolic networks and provides methods for solving the
resulting mixed-integer linear programing problems. The metabolic modeling and simulation
methods referred to herein as OptKnock are described in, for example,
U.S. publication 2002/0168654, filed January 10, 2002, in International Patent No.
PCT/US02/00660, filed January 10, 2002, and
U.S. publication 2009/0047719, filed August 10, 2007.
[0231] Another computational method for identifying and designing metabolic alterations
favoring biosynthetic production of a product is a metabolic modeling and simulation
system termed SimPheny®. This computational method and system is described in, for
example,
U.S. publication 2003/0233218, filed June 14, 2002, and in International Application No.
PCT/US03/18838, filed June 13, 2003. SimPheny® is a computational system that can be used to produce a network model
in silico and to simulate the flux of mass, energy or charge through the chemical reactions
of a biological system to define a solution space that contains any and all possible
functionalities of the chemical reactions in the system, thereby determining a range
of allowed activities for the biological system. This approach is preferred to as
constraints-based modeling because the solution space is defined by constraints such
as the known stoichiometry of the included reactions as well as reaction thermodynamic
and capacity constraints associated with maximum fluxes through reactions. The space
defined by these constraints can be interrogated to determine the phenotypic capabilities
and behavior of the biological system or of its biochemical components.
[0232] These computational approaches are consistent with biological realities because biological
systems are flexible and can reach the same result in many different ways. Biological
systems are designed through evolutionary mechanisms that have been restricted by
fundamental constraints that all living systems must face. Therefore, constraints-based
modeling strategy embraces these general realities. Further, the ability to continuously
impose further restrictions on a network model via the tightening of constraints results
in a reduction in the size of the solution space, thereby enhancing the precision
with which physiological performance or phenotype can be predicted.
[0233] Given the teachings and guidance provided herein, those skilled in the art will be
able to apply various computational frameworks for metabolic modeling and simulation
to design and implement biosynthesis of a desired compound in host microbial organisms.
Such metabolic modeling and simulation methods include, for example, the computational
systems exemplified above as SimPheny® and OptKnock, For illustration of the invention,
some methods are described herein with reference to the OptKnock computation framework
for modeling and simulation. Those skilled in the art will know how to apply the identification,
design and implementation of the metabolic alterations using OptKnock to any of such
other metabolic modeling and simulation computational frameworks and methods well
known in the art.
[0234] The methods described above will provide one set of metabolic reactions to disrupt.
Elimination of each reaction within the set or metabolic modification can result,
in a desired product as an obligatory product during the growth phase of the organism.
Because the reactions are known, a solution to the bilevel OptKnock problem will provide
the associated gene or genes encoding one or more enzymes that catalyze each reaction
within the set of reactions. Identification of a set of reactions and their corresponding
genes encoding the enzymes participating in each reaction is generally an automated
process, accomplished through correlation of the reactions with a reaction database
having a relationship between enzymes and encoding genes.
[0235] Once identified, the set of reactions that are to be disrupted in order to achieve
production of a desired product are implemented in the target cell or organism by
functional disruption of at least one gene encoding each metabolic reaction within
the set. One particularly useful means to achieve functional disruption of the reaction
set is by deletion of each encoding gene. However, in some instances, it can be beneficial
to disrupt the reaction by other genetic aberrations including, for example, mutation,
deletion of regulatory region such as promoters or cis binding sites for regulatory
factors, or by truncation of the coding sequence at any of a number of locations.
These latter aberrations, resulting in less than total deletion of the gene set can
be useful, for example, when rapid assessments of the coupling of a product are desired
or when genetic reversion is less likely to occur.
[0236] To identify additional productive solutions to the above described bilevel OptKnock
problem which lead to further sets of reactions to disrupt or metabolic modifications
that can result in the biosynthesis, including growth-coupled biosynthesis of a desired
product, an optimization method, termed integer cuts, can be implemented. This method
proceeds by iteratively solving the OptKnock problem exemplified above with the incorporation
of an additional constraint referred to as an integer cut at each iteration. Integer
cut constraints effectively prevent the solution procedure from choosing the exact
same set of reactions identified in any previous iteration that obligatorily couples
product biosynthesis to growth. For example, if a previously identified growth-coupled
metabolic modification specifies reactions 1, 2, and 3 for disruption, then the following
constraint prevents the same reactions from being simultaneously considered in subsequent
solutions. The integer cut method is well known in the art and can be found described
in, for example,
Burgard et al., Biotechnol. Prog. 17:791-797 (2001). As with all methods described herein with reference to their use in combination
with the OptKnock computational framework for metabolic modeling and simulation, the
integer cut method of reducing redundancy in iterative computational analysis also
can be applied with other computational frameworks well known in the art including,
for example, SimPheny®.
[0237] The methods exemplified herein allow the construction of cells and organisms that
biosynthetically produce a desired product, including the obligatory coupling of production
of a target biochemical product to growth of the cell or organism engineered to harbor
the identified genetic alterations. Therefore, the computational methods described
herein allow the identification and implementation of metabolic modifications that
are identified by an
in silico method selected from OptKnock or SimPheny®. The set of metabolic modifications can
include, for example, addition of one or more biosynthetic pathway enzymes and/or
functional disruption of one or more metabolic reactions including, for example, disruption
by gene deletion.
[0238] As discussed above, the OptKnock methodology was developed on the premise that mutant
microbial networks can be evolved towards their computationally predicted maximum-growth
phenotypes when subjected to long periods of growth selection. In other words, the
approach leverages an organism's ability to self-optimize under selective pressures.
The OptKnock framework allows for the exhaustive enumeration of gene deletion combinations
that force a coupling between biochemical production and cell growth based on network
stoichiometry. The identification of optimal gene/reaction knockouts requires the
solution of a bilevel optimization problem that chooses the set of active reactions
such that an optimal growth solution for the resulting network overproduces the biochemical
of interest (
Burgard et al., Biotechnol. Bioeng. 84:647-657 (2003)).
[0239] An
in silico stoichiometric model of
E.
coli metabolism can be employed to identify essential genes for metabolic pathways as
exemplified previously and described in, for example, U.S. patent publications
US 2002/0012939,
US 2003/0224363,
US 2004/0029149,
US 2004/0072723,
US 2003/0059792,
US 2002/0168654 and
US 2004/0009466, and in
U.S. Patent No. 7,127,379. As disclosed herein, the OptKnock mathematical framework can be applied to pinpoint
gene deletions leading to the growth-coupled production of a desired product. Further,
the solution of the bilevel OptKnock problem provides only one set of deletions. To
enumerate all meaningful solutions, that is, all sets of knockouts leading to growth-coupled
production formation, an optimization technique, termed integer cuts, can be implemented.
This entails iteratively solving the OptKnock problem with the incorporation of an
additional constraint referred to as an integer cut at each iteration, as discussed
above.
[0240] As disclosed herein, a nucleic acid encoding a desired activity of a toluene, benzene,
p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate,
benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene pathway can be
introduced into a host organism. In some cases, it can be desirable to modify an activity
of a toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate,
(2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol
or 1,3-butadiene pathway enzyme or protein to increase production of toluene, benzene,
p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate,
benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene . For example,
known mutations that increase the activity of a protein or enzyme can be introduced
into an encoding nucleic acid molecule. Additionally, optimidation methods can be
applied to increase the activity of an enzyme or protein and/or decrease an inhibitory
activity, for example, decrease the activity of a negative regulator.
[0241] One such optimization method is directed evolution. Directed evolution is a powerful
approach that involves the introduction of mutations targeted to a specific gene in
order to improve and/or alter the properties of an enzyme. Improved and/or altered
enzymes can be identified through the development and implementation of sensitive
high-throughput screening assays that allow the automated screening of many enzyme
variants (for example, >104). Iterative rounds of mutagenesis and screening typically
are performed to afford an enzyme with optimized properties. Computational algorithms
that can help to identify areas of the gene for mutagenesis also have been developed
and can significantly reduce the number of enzyme variants that need to be generated
and screened. Numerous directed evolution technologies have been developed (for reviews,
see
Hibbert et al., Biomol.Eng 22:11-19 (2005);
Huisman and Lalonde, In Biocatalysis in the pharmaceutical and biotechnology industries
pgs. 717-742 (2007), Patel (ed.), CRC Press;
Otten and Quax. Biomol. Eng 22:1-9 (2005).; and
Sen et al., Appl Biochem.Biotechnol 143:212-223 (2007)) to be effective at creating diverse variant libraries, and these methods have been
successfully applied to the improvement of a wide range of properties across many
enzyme classes. Enzyme characteristics that have been improved and/or altered by directed
evolution technologies include, for example: selectivity/specificity, for conversion
of non-natural substrates; temperature stability, for robust high temperature processing;
pH stability, for bioprocessing under lower or higher pH conditions; substrate or
product tolerance, so that high product titers can be achieved; binding (K
m), including broadening substrate binding to include non-natural substrates; inhibition
(K
i), to remove inhibition by products, substrates, or key intermediates; activity (kcat),
to increases enzymatic reaction rates to achieve desired flux; expression levels,
to increase protein yields and overall pathway flux; oxygen stability, for operation
of air sensitive enzymes under aerobic conditions; and anaerobic activity, for operation
of an aerobic enzyme in the absence of oxygen.
[0242] A number of exemplary methods have been developed for the mutagenesis and diversification
of genes to target desired properties of specific enzymes. Such methods are well known
to those skilled in the art. Any of these can be used to alter and/or optimize the
activity of a toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate,
(2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol
or 1,3-butadiene pathway enzyme or protein. Such methods include, but are not limited
to EpPCR, which introduces random point mutations by reducing the fidelity of DNA
polymerase in PCR reactions (
Pritchard et al., J. Theor.Biol. 234:497-509 (2005)); Error-prone Rolling Circle Amplification (epRCA), which is similar to epPCR except
a whole circular plasmid is used as the template and random 6-mers with exonuclease
resistant thiophosphate linkages on the last 2 nucleotides are used to amplify the
plasmid followed by transformation into cells in which the plasmid is re-circularized
at tandem repeats (
Fujii et al., Nucleic Acids Res. 32:e145 (2004); and
Fujii, et al., Nat. Protoc. 1:2493-2497 (2006)); DNA or Family Shuffling, which typically involves digestion of two or more variant
genes with nucleases such as Dnase I or EndoV to generate a pool of random fragments
that are reassembled by cycles of annealing and extension in the presence of DNA polymerase
to create a library of chimeric genes (
Stemmer, Proc. Natl. Acad. Sci. U.S.A. 91:10747-10751 (1994); and
Stemmer, Nature 370:389-391 (1994)); Staggered Extension (StEP), which entails template priming followed by repeated
cycles of 2 step PCR with denaturation and very short duration of annealing/extension
(as short as 5 sec) (
Zhao et al., Nat. Biotechnol. 16:258-261 (1998)); Random Priming Recombination (RPR), in which random sequence primers are used
to generate many short DNA fragments complementary to different segments of the template
(
Shao et al., Nucleic Acids Res 26:681-683 (1998)).
[0243] Additional methods include Heteroduplex Recombination, in which linearized plasmid
DNA is used to form heteroduplexes that are repaired by mismatch repair (
Volkov et al, Nucleic Acids Res. 27:e18 (1999); and
Volkov et al., Methods Enzymol. 328:456-463 (2000)); Random Chimeragenesis on Transient Templates (RACHITT), which employs Dnase I
fragmentation and size fractionation of single stranded DNA (ssDNA) (
Coco et al., Nat. Biotechnol. 19:354-359 (2001)); Recombined Extension on Truncated templates (RETT), which entails template switching
of unidirectionally growing strands from primers in the presence of unidirectional
ssDNA fragments used as a pool of templates (
Lee et al., J. Molec. Catalysis 26:119-129 (2003)); Degenerate Oligonucleotide Gene Shuffling (DOGS), in which degenerate primers
are used to control recombination between molecules; (
Bergquist and Gibbs, Methods Mol. Biol. 352:191-204 (2007);
Bergquist et al., Biomol. Eng. 22:63-72 (2005);
Gibbs et al., Gene 271:13-20 (2001)); Incremental Truncation for the Creation of Hybrid Enzymes (ITCHY), which creates
a combinatorial library with 1 base pair deletions of a gene or gene fragment of interest
(
Ostermeier et al., Proc. Natl. Acad. Sci. U.S.A. 96:3562-3567 (1999); and
Ostermeier et al., Nat. Biotechnol. 17:1205-1209 (1999)); Thio-Incremental Truncation for the Creation of Hybrid Enzymes (THIO-ITCHY), which
is similar to ITCHY except that phosphothioate dNTPs are used to generate truncations
(
Lutz et al., Nucleic Acids Res. 29:E16 (2001)); SCRATCHY, which combines two methods for recombining genes, ITCHY and DNA shuffling
(
Lutz et al., Proc. Natl. Acad. Sci. U.S.A. 98:11248-11253 (2001)); Random Drift Mutagenesis (RNDM), in which mutations made via epPCR are followed
by screening/selection for those retaining usable activity (
Bergquist et al., Biomol. Eng. 22:63-72 (2005)); Sequence Saturation Mutagenesis (SeSaM), a random mutagenesis method that generates
a pool of random length fragments using random incorporation of a phosphothioate nucleotide
and cleavage, which is used as a template to extend in the presence of "universal"
bases such as inosine, and replication of an inosine-containing complement gives random
base incorporation and, consequently, mutagenesis (
Wong et al., Biotechnol. J. 3:74-82 (2008);
Wong et al., Nucleic Acids Res. 32:e26 (2004); and
Wong et al., Anal. Biochem. 341:187-189 (2005)); Synthetic Shuffling, which uses overlapping oligonucleotides designed to encode
"all genetic diversity in targets" and allows a very high diversity for the shuffled
progeny (
Ness et al., Nat. Biotechnol. 20:1251-1255 (2002)); Nucleotide Exchange and Excision Technology NexT, which exploits a combination
of dUTP incorporation followed by treatment with uracil DNA glycosylase and then piperidine
to perform endpoint DNA fragmentation (
Muller et al., Nucleic Acids Res. 33:e117 (2005)).
[0244] Further methods include Sequence Homology-Independent Protein Recombination (SHIPREC),
in which a linker is used to facilitate fusion between two distantly related or unrelated
genes, and a range of chimeras is generated between the two genes, resulting in libraries
of single-crossover hybrids (
Sieber et al., Nat. Biotechnol. 19:456-460 (2001)); Gene Site Saturation Mutagenesis™ (CSSM™), in which the starting materials include
a supercoiled double stranded DNA (dsDNA) plasmid containing an insert and two primers
which are degenerate at the desired site of mutations (
Kretz et al., Methods Enzymol. 388:3-11 (2004)); Combinatorial Cassette Mutagenesis (CCM), which involves the use of short oligonucleotide
cassettes to replace limited regions with a large number of possible amino acid sequence
alterations (
Reidhaar-Olson et al. Methods Enzymol. 208:564-586 (1991); and
Reidhaar-Olson et al. Science 241:53-57 (1988)); Combinatorial Multiple Cassette Mutagenesis (CMCM), which is essentially similar
to CCM and uses epPCR at high mutation rate to identify hot spots and hot regions
and then extension by CMCM to cover a defined region of protein sequence space (
Reetz et al., Angew. Chem. Int. Ed Engl. 40:3589-3591 (2001)); the Mutator Strains technique, in which conditional ts mutator plasmids, utilizing
the mutD5 gene, which encodes a mutant subunit of DNA polymerase III, to allow increases
of 20 to 4000-X in random and natural mutation frequency during selection and block
accumulation of deleterious mutations when selection is not required (
Selifonova et al., Appl. Environ. Microbiol. 67:3645-3649 (2001));
Low et al., J. Mol. Biol. 260:359-3680 (1996)).
[0245] Additional exemplary methods include Look-Through Mutagenesis (LTM), which is a multidimensional
mutagenesis method that assesses and optimizes combinatorial mutations of selected
amino acids (
Rajpal et al., Proc. Natl. Acad. Sci. U.S.A. 102:8466-8471 (2005)); Gene Reassembly, which is a DNA shuffling method that can be applied to multiple
genes at one time or to create a large library of chimeras (multiple mutations) of
a single gene (Tunable GeneReassembly™ (TGR™) Technology supplied by Verenium Corporation),
in Silico Protein Design Automation (PDA), which is an optimization algorithm that
anchors the structurally defined protein backbone possessing a particular fold, and
searches sequence .for acid substitutions that can stabilize the fold and overall
protein energetics, and generally works most effectively on proteins with known three-dimensional
structures (
Hayes et al., Proc. Natl. Acad. Sci. U.S.A. 99:15926-15931 (2002)); and Iterative Saturation Mutagenesis (ISM), which involves using knowledge of
structure/function to choose a likely site for enzyme improvement, performing saturation
mutagenesis at chosen site using a mutagenesis method such as Stratagene QuikChange
(Stratagene; San Diego CA), screening/selecting for desired properties, and, using
improved clone(s), starting over at another site and continue repeating until a desired
activity is achieved (
Reetz et al., Nat. Protoc. 2:891-903 (2007); and
Reetz et al., Angew. Chem. Int. Ed Engl. 45:7745-7751 (2006)).
[0246] Any of the aforementioned methods for mutagenesis can be used alone or in any combination.
Additionally, any one or combination of the directed evolution methods can be used
in conjunction with adaptive evolution techniques, as described herein.
EXAMPLE I
Pathways to Benzene and Toluene
[0247] This example shows pathways from phenylalanine to toluene, phenylalanine to benzene
and benzoyl-CoA to styrene.
[0248] Pathways for enzymatic conversion of phenylalanine are shown in Figure 1. The first
step entails conversion of phenylalanine to phenylpyruvate, a transformation that
can be accomplished by an aminotransferase or a deaminating oxidoreductase. Phenylpyruvate
is then decarboxylated to phenylacetaldehyde in Step B of Figure 1. Toluene may be
produced directly from phenylacetaldehyde by decarbonylation (Figure 1, Step E), or
indirectly via a phenylacetate intermediate (Figure 1, Steps C and D). Phenylacetate
can be oxidized to phenylacetate (Figure 1, Step C) by either a phenylacetaldehyde
dehydrogenase or a phenylacetaldehyde oxidase. An alternate pathway is the direct
oxidative decarboxylation of phenylpyruvate to phenylacetate by phenylpyruvate oxidase
(Figure 1, StepF).
[0249] A one-step pathway for enzymatic conversion of phenylalanine to benzene is shown
in Figure 2. The conversion of phenylalanine and water to benzene, pyruvate and ammonia
is catalyzed by an enzyme with phenylalanine benzene-lyase activity.
[0250] Enzymatic pathways to styrene from benzoyl-CoA are shown in Figure 3. Benzoyl-CoA
is a common metabolic intermediate of numerous biosynthetic and degradation pathways.
Pathways involving the biosynthesis of benzoyl-CoA, and also the generation of benzoyl-CoA
as a degradation product, are known in the art. In the proposed styrene pathways,
benzoyl-CoA and acetyl-CoA are first converted to 3-oxo-3-phenylpropionyl-CoA by a
beta-ketothiolase (Figure 3, Step A). The CoA moiety of 3-oxo-3-phenylpropionyl-CoA
is then released by a CoA hydrolase, transferase or synthase (Figure 3, Step B). Alternately,
3-oxo-3-phenylpropionyl-CoA is converted to benzoyl-acetate in two enzymatic steps
by a phosphotrans-3-oxo-3-phenylpropionylase and benzoyl-acetate kinase (Figure 3,
Steps F and G). Once formed, benzoyl-acetate is decarboxylated, reduced and dehydrated
to form styrene (Figure 3, Steps C, D and E).
[0251] Enzymes for catalyzing the transformations shown in Figures 1-3 are categorized by
EC number (Table 1) and described further below.
Label |
Function |
Step |
1.1.1.a |
Oxidoreductase (oxo to alcohol) |
3D |
1.2.1.a |
Oxidoreductase (aldehyde to acid) |
1C |
1.2.3.a |
Aldehyde oxidase |
1C/F |
1.4.1.a |
Oxidoreductase (aminating/deaminating) |
1A |
2.3.1.a |
Acyltransferase (transferring phosphate group to CoA) |
3F |
2.3.1.b |
Beta-ketothiolase |
3A |
2.6.1.a |
Amminotransferase |
1A |
2.7.2.a |
Phosphotransferase, carboxyl group acceptor (kinase) |
3G |
2.8.3.a |
Coenzyme-A transferase |
3B |
3.1.2.a |
Thiolester hydrolase (CoA specific) |
3B |
4.1.1.a |
Carboxy-lyase |
1B/D, 3 C |
4.1.99.a |
Decarbonylase |
1E |
4.1.99.b |
Lyase |
2 |
4.2.1.a |
Hydro-lyase |
3E |
6.2.1.a |
Acid-thiol ligase |
3B |
[0252] 1.1.1.a Oxidoreductase (oxo to alcohol): The reduction of acetophenone to 1-phenylethanol
(
Step D of Figure 3) is catalyzed by a ketone reductase with acetophenone reductase activity. Enzymes
with this activity have been characterized in numerous organisms, and product formation
is generally stereoselective. An exemplary enzyme with this activity is the R-specific
short-chain dehydrogenase/reductase of
Lactobacillus brevis, which has been extensively studied, structurally characterized and re-engineered
to prefer NADH to NADPH as a cosubstrate (
Schlieben et al., J. Mol.Biol. 349:801-813 (2005)). Additional enzymes with acetophenone reductase activity are encoded by
adhF1 of
Pseudomonas fluorescens (
Hildebrandt et al., Appl. Microbiol Biotechnol. 59:483-487 (2002)),
adh of
Thermus thermophilus (
Pennacchio et al., Appl. Environ. Microbiol 74:3949-3958 (2008)) and
LSADH of
Leifsonia sp. S749 (
Inoue et al., Biosci . Biotechnol. Biochem. 70:418-426 (2006)). An S-specific enzyme was characterized in the ethylbenzene degradation pathway
of the denitrifying bacterium
Aromatoleum aromaticum EbN1 (
Kniemeyer et al., Arch. Microbiol. 176:129-135 (2001)). This enzyme, encoded by
ped, favors the reductive direction at low pH (4), while the oxidative direction is favored
at neutral pH.
Gene |
GenBank Accession No. |
GI No. |
Organism |
LVIS_0347 |
YP_794544.1 |
116333017 |
Lactobacillus brevis |
adhF1 |
AAL79772.1 |
18860822 |
Pseudomonas fluorescens |
adhTt |
YP_003977.1 |
46198310 |
Thermus thermophilus |
LSADH |
BAD99642.1 |
67625613 |
Leifsonia sp. 5749 |
ped |
YP_58329.1 |
56476740 |
Aromatoleum aromaticum EbN1 |
[0253] A variety of alcohol dehydrogenase enzymes catalyze the reduction of a ketone to
an alcohol functional group. These enzymes are also suitable for catalyzing the reduction
of acetophenone. Two such enzymes in
E. coli are encoded by malate dehydrogenase (
mdh) and lactate dehydrogenase (
ldhA). The lactate dehydrogenase from
Ralstonia eutropha has been shown to demonstrate high activities on 2-ketoacids of various chain lengths
including lactate, 2-oxobutyrate, 2-oxopentanoate and 2-oxoglutarate (
Steinbuchel et al., Eur. J. Biochem. 130:329-334 (1983)). Conversion of alpha-ketoadipate into alpha-hydroxyadipate can be catalyzed by
2-ketoadipate reductase, an enzyme reported to be found in rat and in human placenta
(
Suda et al., Arch. Biochem. Biophys. 176:610-620 (1976);
Suda et al., Biochem. Biophys. Res. Commun. 77:586-591 (1977)). An additional oxidoreductase is the mitochondrial 3-hydroxybutyrate dehydrogenase
(
bdh) from the human heart which has been cloned and characterized (
Marks et al., J. Biol. Chem. 267:15459-15463 (1992)). Alcohol dehydrogenase enzymes of
C.
beijerinckii (
Ismaiel et al., J.Bacteriol. 175:5097-5105 (1993)) and
T. brockii (
Lamed et al., Biochem. J. 195:183-190 (1981);
Peretz et al., Biochemistry 28:6549-6555 (1989)) convert acetone to isopropanol. Methyl ethyl ketone reductase, or alternatively,
2-butanol dehydrogenase, catalyzes the reduction of MEK to form 2-butanol. Exemplary
MEK reductase enzymes can be found in
Rhodococcus ruber (
Kosjek et al., Biotechnol Bioeng. 86:55-62 (2004)) and
Pyrococcus furiosus (
van der et al., Eur. J. Biochem. 268:3062-3068 (2001)).
Gene |
GenBank Accession No. |
GI No. |
Organism |
Mdh |
AAC76268.1 |
1789632 |
Escherichia coli |
ldhA |
NP_415898.1 |
16129341 |
Escherichia coli |
Ldh |
YP_725182.1 |
113866693 |
Ralstonia eutropha |
bdh |
AAA58352.1 |
177198 |
Homo sapiens |
adh |
AAA23199.2 |
60592974 |
Clostridium beijerinckii NRRL B593 |
adh |
P14941.1 |
113443 |
Thermoanaerobacter broakii HTD4 |
adhA |
AAC25556 |
3288810 |
Pyrococcus furiosus |
sadh |
C4D36473 |
21615553 |
Rhodococcus ruber |
[0254] 1.2.1.a Oxidoreductase (aldehyde to acid): Oxidation of phenylacetaldehyde to phenylacetate
is catalyzed by phenylacetaldehyde dehydrogenase
(Step C of Figure 1), an enzyme in the EC class 1.2.1. NAD
+-dependent phenylacetaldehyde dehydrogenase enzymes (EC 1.2.1.39) have been characterized
in
E. coli, Pseudomonas putida and Antirrhinum majus (
Long et al., Plant J 59:256-265 (2009); et al.,
Environ.Microbiol 10:413-432 (2008);
Ferrandez et al., FEBS Lett. 406:23-27 (1997)). NAD
+-dependent aldehyde dehydrogenase enzymes with high activity on phenylacetaldehyde
have also been characterized in mammals such as
Bos taurus, Rattus norvegicus, and
Homo sapiens (
Klyosov, Biochemistry 35:4457-4467 (1996a);
Lindahl et al., J Biol.Chem. 259:11991-11996 (1984)). Two aldehyde dehydrogenases found in human liver, ALDH-1 and ALDH-2, have broad
substrate ranges for a variety of aliphatic, aromatic and polycyclic aldehydes with
demonstrated activity on phenylacetaldehyde (
Klyosov, Biochemistry 35:4457-4467 (1996b)). The NADP
+-dependent benzaldehyde dehydrogenase of
Pseudomonas putida encoded by
badh also demonstrated activity on phenylacetaldehyde (
Yeung et al., Biochim.Biophys.Acta 1784:1248-1255 (2008)). NAD
+-dependent aldehyde dehydrogenase enzymes with high activity on phenylacetaldehyde
have also been characterized in mammals such as
Bos taurus, Rattus norvegicus and
Homo sapiens (
Klyosov, Biochemistry 35:4457-4467 (1996a);
Lindahl et al., J Biol.Chem. 259:11991-11996 (1984)). Two aldehyde dehydrogenases found in human liver, ALDH-1 and ALDH-2, have broad
substrate ranges for a variety of aliphatic, aromatic and polycyclic aldehydes with
demonstrated activity on phenylacetaldehyde (
Klyosov, Biochemistry 35:4457-4467 (1996b)).
Gene |
GenBank Accession No. |
GI No. |
Organism |
feaB |
AAC74467.2 |
87081896 |
Escherichia coli |
peaE |
ABR57205.1 |
150014683 |
Pseudomonas putida |
BALDH |
ACM89738.1 |
223452696 |
Antirrhinum majus |
ALDH-2 |
P05091.2 |
118504 |
Homo sapiens |
badh |
P39849.1 |
731175 |
Pseudomonas putida |
[0255] 1.2.3.a Aldehyde oxidase: An O
2-dependent aldehyde oxidase enzyme can be employed to convert phenylacetaldehyde or
phenylpyruvate to phenylacetate (Steps C and F of Figure 1). Phenylacetaldehyde oxidase
enzymes convert phenylacetaldehyde, water and O
2 to phenylacetate and hydrogen peroxide. Exemplary phenylacetaldehyde oxidase enzymes
are found in
Methylobacillus sp., Pseudomonas sp., Streptomyces moderatus,
Cavia porcellus and
Zea mays (
Koshiba et al., Plant Physiol 110:-781-789 (1996)). The two flavin- and molybdenum- containing aldehyde oxidases of
Zea mays are encoded by
zmAO-1 and
zmAO-2 (
Sekimoto et al., J Biol.Chem. 272:15280-15285 (1997)). Phenylacetaldehyde oxidase activity has also been demonstrated in the indole-3-acetaidehyde
oxidase (EC 1.2.3.7) of
Avena sativa, although the genes associated with this activity have not been identified to date
and bear no significant homology to the aldehyde oxidase genes of
Zea mays (
Rajagopal, Physicol.Plant 24:272-281 (1971)). Additional phenylacetaldehyde oxidases can be inferred by sequence homology to
the
Z.
mays genes and are shown below.
Gene |
GenBank Accession No. |
GI No. |
Organism |
zmAO-1 |
NP_001105308.1 |
162458742 |
Zea mays |
zmAO-2 |
BAA23227.1 |
2589164 |
Zea mays |
Aox1 |
054754.2 |
20978408 |
Mus musculus |
ALDO1_ORYSJ |
Q7XH05.1 |
75296231 |
Oryza sativa |
AAO3 |
BAA82672.1 |
5672672 |
Arabidopsis thaliana |
XDH |
DAA24801.1 |
296482686 |
Bos taurus |
[0256] Phenylpyruvate oxidase enzymes convert, phenylpyruvate and O2 to phenylacetate, CO2
and water (Step F of Figure 1). The 4-hydroxyphenylpyruvate oxidase (EC 1.2.3.13)
from Arthrobacter globiformis was shown catalyze the O2-dependent oxidation of phenylpyruvate
to phenylacetate during tyrosine catabolism (
Blakley, Can.J Microbial 23:1128-1139 (1977)). This enzymatic activity was demonstrated in cell extracts; however, the gene encoding
this enzyme has not been identified to date.
[0257] 1.4.1.a. Oxidoreductase (deaminating): The NAD(P)+-dependent oxidation of phenylalanine
to phenylpyravate (Step A of Figure 1) is catalyzed by phenylalanine oxidoreductase
(deaminating), also called phenylalanine dehydrogenase. NALD+-dependent phenylalanine
dehydrogenase enzymes encoded by pdh genes have been characterized in Bacillus
badius,
Lysinibacillus sphaericus and
Thermoactinotinomyces intermedius (
Yamada et al., Biosci.Biotechnol.Biochem. 59:1994-1995 (1995);
Takada et al., J Biochem. 109:371-376 (1991);
Okazaki et al., Gene 63:337-341 (1988)).
Gene |
GenBank Accession No. |
GI No. |
Organism |
pdh |
BAA08816.1 |
1228936 |
Bacillus badius |
pdh |
AAA22646.1 |
529017 |
Lysinibacillus sphaericus |
pdh |
P22823.1 |
118598 |
Thermoactinomyces intermedius |
[0258] 2.3.1.a. Acyltransferase (phosphotransacylase): An enzyme with phosphotransbenzoylase
activity is required to phosphorylate 3-oxo-3-phenylpropionyl-CoA to [(3-oxo-3-phenylpropionyl)oxy]phosphoate
Step F of Figure 3). An enzyme with this activity has not been characterized to date. Exemplary phosphate-transferring
acyltransferases include phosphotransacetylase (EC 2.3.1.8) and phosphotransbutyrylase
(EC 2.3.1.19). The
pta gene from
E. coli encodes a phosphotransacetylase that reversibly converts acetyl-CoA into acetyt-phosphate
(
Suzuki, Biochimi.Biophys.Acta 191:559-569 (1969)). Phosphotransacetylase enzymes in several organisms also catalyze the conversion
of propionyl-CoA to propionylphosphate. Such enzymes include the
pta gene products of
E. coli (
Hesslinger et al., Mol.Microbiol 27:477-492 (1998)),
Bacillus subtilis (
Rado et al., Biochim.Biophys.Acta 321:114-125 (1973)),
Clostridium kluyveri (Stadtman, 1:596-599 (1955)), and
Thermotoga maritima (
Bock et al., J Bacteriol). 181:1861-1867 (1999)). The
ptb gene from
C. acetobutylicum encodes phosphotransbutyrylase, an enzyme that reversible converts butyryt-CoA into
butyryl-phosphate (
Wiesenborn et al., Appl Environ.Microbiol 55:317-322 (1989);
Walter et al., Gene 134:107-111 (1993)). Additional
ptb genes are found in butyrate-producing bacterium L2-50 (
Louis et al., J.Bacteriol. 186:2099-2106 (2004)) and
Bacillus migaterium (
Vazquez et al., Curr.Microbiol 42:345-349 (2001)).
Gene |
GenBank Accession No. |
GI No. |
Organism |
pta |
NP_416800.1 |
71152910 |
Escherichia coli |
pta |
P39646 |
730415 |
Bacillus subtilis |
pta |
A5N801 |
146346896 |
Clostridium kluyveri |
pta |
Q9X0L4 |
6685776 |
Thermotoga maritime |
ptb |
NP_349676 |
34540484 |
Clostridium acetobutylicum |
ptb |
AAR.19757.1 |
38425288 |
butyrate-producing bacterium L2-50 |
ptb |
CAC07932.1 |
10046659 |
Bacillus megaterium |
[0259] 2.3.1.b Beta-ketothiolase. A beta-ketothiolase enzyme is required to convert benzoyl-CoA
and acetyl-CoA to 3-oxo-3-phenylpropionyl-CoA (Step A of Figure 3). This transformation
is not known to occur naturally. Suitable beta-ketothiolase enzymes include 3-oxoadipyl-CoA
thiolase (EC 2.3.1.174), 3-oxopimeloyl-CoA:glutaryl-CoA acyltransferase (EC 2.3.1.16),
3-oxovaleryl-CoA thiolase and aectoacetyl-CoA thiolase (EC 2.1.3.9). 3-Oxoadipyl-CoA
thiolase (EC 2.3.1.174) converts beta-ketoadipyl-CoA to succinyl-CoA and acetyl-CoA,
and is a key enzyme of the beta-ketoadipate pathway for aromatic compound degradation.
The enzyme is widespread in soil bacteria and fungi including Pseudomonas putida (
Harwood et al., J Bacteriol. 176:6479-6488 (1994)) and Acinetobacter calcoaceticus (
Doten et al., J Bacteriol. 169:3168-3174 (1987)). The gene products encoded by pcaF in Pseudomonas strain B13 (
Kaschabek et al., J Bacteriol. 184:207-215 (2002)), phaD in Pseudomonas putida U (
Olivera et al., Proc.Natl.Acad.Sci U.S.A 95:6419-6424 (1998)), paaE in Pseudomonas fluorescens ST (
Di et al., Arch.Microbiol 188:117-125 (2007)), and paaJ from E. coli (
Nogales et al., Microbiology 153:357-365 (2007)) also catalyze this transformation. Several beta-ketothiolases exhibit significant
and selective activities in the oxoadipyl-CoA forming direction including
bkt from
Pseudomonas putida,
pcaF and
bkt from
Pseudomonas aeruginosa PAO1, bkt from
Burkholderia ambifaria AMMD,
paaJ from
E.
coli, and
phaD from
P. putida.
Gene name |
GI# |
GenBank Accession # |
Organism |
paaJ |
16129358 |
NP_415915.1 |
Escherichia coli |
pcaF |
17736947 |
AAL02407 |
Pseudomonas knackmussii (B13) |
phaD |
3253200 |
AAC24332.1 |
Pseudomonas putida |
pcaF |
506695 |
AAA85138.1 |
Pseudomonas putida |
pcaF |
141777 |
AAC37148.1 |
Acinetobacter calcoaceticus |
paaE |
106636097 |
ABF82237.1 |
Pseudomonas fluorescens |
bkt |
115360515 |
YP_777652.1 |
Burkholderia ambifaria AMMD |
bkt |
9949744 |
AAG06977.1 |
Pseudomonas aeruginosa PAO1 |
pcaF |
9946065 |
AAG03617.1 |
PSeudomonas aeruginosa PAO1 |
[0260] 3-Oxopimeloyl-CoA thiolase catalyzes the condensation of glutaryl-CoA and acetyl-CoA
into 3-oxopimeloyl-CoA (EC 2.3.1.16). An enzyme catalyzing this transformation is
encoded by genes
bktB and
bktc in Ralstonia eutropha (formerly known as
Alcaligenes eutrophus) (
Slater et al., J.Bacteriol. 180:1979-1987 (1998);
Haywood et al., FEMS Microbiology Letters 52:91-96 (1988)). The sequence of the BktB protein is known but the sequence of the BktC protein
has not been reported to date. The
pim operon of Rhodopseudomonas
palustris also encodes a beta-ketothiolase, encoded by
pimB, predicted to catalyze this transformation in the degradative direction during benzoyl-COA
degradation (
Harrison et al., Microbiology 151:727-736 (2005)). A beta-ketothiolase enzyme in.
S.
aciditrophicus was identified by sequence homology to
bktB (43% identity, evalue = 1e-93).
Gene name |
GI# |
GenBank Accession # |
Organism |
bktB |
11386745 |
YP_725948 |
Ralstonia eutropha |
pimB |
39650633 |
CAE29156 |
Rhodopseudomonas palustris |
syn_02642 |
85860483 |
YP_462685.1 |
Syntrophus aciditrophicus |
[0261] Beta-ketothiolase enzymes catalyzing the formation of 3-oxovalerate from acetyl-CoA
and propionyl-CoA may also be to catalyze the formation of 3-oxo-3-phenylpropionyl-CoA.
Zoogloea ramigera possesses two ketothiolases that can form β-ketovaletyl-CoA from propionyl-CoA and
acetyl-CoA and
R. eutropha has a β -oxidation ketothiolase that is also capable of catalyzing this transformation
(Gruys et al., (1999)). The sequences of these genes or their translated proteins
have not been reported, but several genes in
R. eutropha, Z.
ramigera, or other organisms can be identified based on sequence homology to
bktB from
R. eutropha and are listed below.
Gene name |
GI# |
GenBank Accession # |
Organism |
phaA |
113867452 |
YP_725941.1 |
Ralstonia eutropha |
h16_A1713 |
113867716 |
YP_726205.1 |
Ralstonia eutropha |
pcaF |
116694155 |
YP_728366.1 |
Ralstonia eutropha |
h16_B1369 |
116695312 |
YP_840888.1 |
Ralstonia eutropha |
h16-A0170 |
113866201 |
YP_724690.1 |
Ralstonia eutropha |
h16_A0462 |
113866491 |
YP_724980.1 |
Ralstonia eutropha |
h16_A1528 |
113867539 |
YP_726028.1 |
Ralstonia eutropha |
h16_B0381 |
116694334 |
YP_728545.1 |
Ralstonia eutropha |
h16_B08062 |
116694613 |
YP_728824.1 |
Ralstonia eutropha |
h16_B0759 |
116694710 |
YP_728921.1 |
Ralstonia eutropha |
h16_80668 |
116694019 |
YP_728830.1 |
Ralstonia eutropha |
h16_A1720 |
113867723 |
YP_726212.1 |
Ralstonia eutropha |
h16_A1887 |
113867867 |
YP_726356.1 |
Ralstonia eutrolpha |
phbA |
135759 |
P07097.4 |
Zoogloea ramigera |
bktB |
194289475 |
YP_002005382.1 |
Cupriavidus taiwanensis |
Rmet_1362 |
94310304 |
YP_583514.1 |
Raltonia metallidurans |
Bphy_0975 |
186475740 |
YP_001857210.1 |
Burkholderia phymatum |
[0263] 2.6.1.a Aminotransferase: A phenylalanine aminotransferase or transaminase in the
EC class 2.6.1 is required to convert phenylalanine to phenylpyruvate
(Step A of Figure 1). A variety of enzymes catalyze this transformation, including phenylacetate aminotransferase,
aromatic amino acid aminotransferase, tryptophan aminotransferase, aspartate aminotransferase,
branched chain amino acid aminotransferase and others. Enzymes with pbenylalanine
aminotransferase activity in
E. coli are encoded by
tyrB, ilvE and
aspC (
Lee-Peng et al., J Bacteriol. 139:339-345 (1979);
Powell et al., Eur.J Biochem. 87:391-400 (1978)). Exemplary enzymes with phenylalanine aminotransferase activity in other organisms
include the aromatic amino acid aminotransferase of
S.
cerevisiae encoded by
AR09 (
Iraqui et al., Mol.Gen.Genet. 257:238-248 (1998)) and the tryptophan aminotransferase of
Arabidopsis thaliana, encoded by
TAA1 (
Tao et al., Cell 133:164-176 (2008)).
Gene |
GenBank Accession No. |
GI No. |
Organism |
tyrB |
AAC77024.1 |
1790488 |
Escherichia coli |
ilvE |
AAT48207.1 |
48994963 |
Escherichia coli |
aspC |
NP_415448.1 |
16128895 |
Escherichia coli |
ARO9 |
NP_012005.1 |
6321929 |
Saccharomyces cerevisiae |
TAA1 |
NP_177213.1 |
15223183 |
Arabidopsis thaliana |
[0264] 2.7.2.a Phosphotransferase: Phosphotransferase enzymes in the EC class 2.7.2 convert
carboxylic acids to phosphonic acids with concurrent hydrolysis of one ATP. In
Step G_of Figure 3, a phosphotransferase is required to convert [(3-oxy-3-phenylpropionyl)oxy]phosphonate
and ADP to benzoyl-acetate and ATP. An enzyme with this exact activity has not been
characterized to date. Exemplary enzymes include butyrate kinase (EC 2.7.2.7), isobutyrate
kinase (EC 2.7.2.14), aspartokinase (EC 2.7.2.4), acetate kinase (EC 2.7.2.1) and
gamma-glutamyl kinase (EC 2.7.2.11). Aspartokinase catalyzes the ATP-dependent phosphorylation
of aspartate and participates in the synthesis of several amino acids. The aspartokinase
III enzyme in
E. coli, encoded by
lysC, has a broad substrate range that includes the aromatic compound aspartic acid 1-benzyl
ester, and the catalytic residues involved in substrate specificity have been elucidated
(
Keng et al., Arch. Biochem.Biophys. 335:73-8 (1996)). Two additional kinases in
E. coli include acetate kinase and gamma-glutamyl kinase. The
E. coli acetate kinase, encoded by
ackA (
Skarstedt et al., J.Biol.Chem. 251:6775-6783 (1976)), phosphorylates propionate in addition to acetate (
Hesslinger et al., Mol.Microbiol 27:477-492 (1998)). The
E. coli gamma-glutamyl kinase, encoded by
proB (
Smith et al., J.Bacteriol. 157:545-551 (1984)), phosphorylates the gamma carbonic acid group of glutamate. Butyrate kinase carries
out the reversible conversion of butyryl-phosphate to butyrate during acidogenesis
in
C.
acetobutylicum (
Cary et al., AppL.Environ.Microbiol 56:1576-1.583 (1990)). This enzyme is encoded by either of the two
buk gene products (
Huang et al., J Mol.Microbiol Biotechnol 2:33-38 (2000)). Other butyrate kinase enzymes are found in
C. butyricium and
C.
tetanomorphum (
TWAROG et al., J Bacteriol. 86:112-117 (1963)). A related enzyme, isobutyrate kinase from
Thermotoga maritime, was expressed in
E. coli and crystallized (
Diao et al., J Bacteriol. 191:2521-2529 (2009);
Diao et al., Acta Crystallogr.D.Biol.Crystallogr. 59:1100-1.102 (2003)).
Gene name |
GI# |
GenBank Accession # |
Organism |
lysC |
16131850 |
NP-418448.1 |
Escherichia coli |
ackA |
16130231 |
NP_416799.1 |
Escherichia coli |
proB |
16128228 |
NP_414777.1 |
Escherichia coli |
buk1 |
15896326 |
NP_349675 |
Clostridium acetobutylicum |
buk2 |
20137415 |
Q97II1 |
Clostridium acetobutylicum |
buk2 |
6685256 |
Q9X278.1 |
Thermotoga maritima |
[0265] 2.8.3.a CoA transferase: CoA transferases catalyze the reversible transfer of a CoA
moiety from one molecule to another. Step B of Figure 3 is catalyzed by an enzyme
with 3-oxo-3-phenylpropionyl-CoA transferase activity. In this transformation, benzoyl-acetate
is formed from 3-oxo-3-phenylpropionyl-CoA by the transfer of the CoA to a CoA acceptor
such as acetate, succinate or others. Exemplary CoA transferase enzymes that react
with similar substrates include cinnamoyl-CoA transferase (EC 2.8.3.17) and benzylsuccinyl-CoA
transferase. Cinnamoyl-CoA transferase, encoded by
fldA in
Clostridium sporogenes, transfers a CoA moiety from cinnamoyl-CoA to a variety of aromatic substrates including
phenylacetate, 3-phenylpropionate and 4-phenylbutyrate (
Dickert et al., Eur. J Biochem. 267:3874-3884 (2000)). Benzylsuccinyl-CoA transferase utilizes succinate or maleate as the CoA acceptor,
forming benzylsuccinate from benzylsuccinyl-CoA. This enzyme was characterized in
the reverse direction in denitrifying bacteria
Thauera aromatica, where it is encoded by
bbsEF (
Leutwein et al., J Bacteriol. 183:4289-4295 (2001)).
Gene |
GenBank Accession No. |
GI No. |
Organism |
fldA |
AAL18808.1 |
16417587 |
Clostridium sporogenes |
bbsE |
AAF89840.1 |
9622535 |
Thauera aromatica |
bbsF |
AAF89841.1 |
9622536 |
Thauera aromatica |
[0266] Additional CoA transferase enzymes with diverse substrate ranges include succinyl-CoA
transferase, 4-hydroxybutyryl-CoA transferase, butyryl-CoA transferase, glutaconyl-CoA
transferase and acetoacetyl-CoA transferase. The gene products of
cat1, cat2, and
cat3 of
Clostridium kluyveri have been shown to exhibit succinyl-CoA, 4-hydroxybutyryl-CoA, and butyryl-CoA transferase
activity, respectively (
Seedorf et al., Proc.Natl.Acad.Sci U.S.A 105:2128-2133 (2008);
Sohling et al., J Bacteriol. 178:871-880 (1996)). Similar CoA transferase activities are also present in
Trichomonas vaginalis (
van Grinsven et al., J.Bio/. Chem. 283:1411-1418 (2008)) and
Trypanosoma brucei (
Riviere et al., J.Bio/. Chem. 279:45337-45346 (2004)). The glutaconyl-CoA-transferase (EC 2.8.3.12) from the anaerobic bacterium
Acidaminococcus fermentans reacts with gtutaconyl-CoA. and 3-butenoyl-CoA (
Mack et al., Eur.J.Biochem. 226-41-51 (1994)). The genes encoding this enzyme are
gctA and
gctB. This enzyme exhibits reduced but detectable activity with several alternate substrates
including glutaryl-CoA, 2-hydroxyglutaryl-CoA, adipyl-CoA, crotonyl-CoA and acrylyl-CoA
(
Buckel et al., Eur.J Biochem. 118:315-321 (1981)). The enzyme has been cloned and expressed in
E. coli (
Mack et al.., Eur.J.Biochem. 226:41-51 (1994)). Glutaconate CoA-transferase activity has also been detected in
Clostridium sporosphaeroides and
Clostridium symbiosum. Acetoacetyl-CoA transferase utilizes acetyl-CoA as the CoA donor. This enzyme is
encoded by the
E. coli atoA (alpha subunit) and
atoD (beta subunit) genes (
Korolev et al., Acta Crystallogr.D.Biol.Crystallogr. 58:2116-2121 (2002);
Vanderwinkel et al., Biochem.Biophys.Res.Commun. 33:902-908 (1968)). This enzyme has a broad substrate range (
Sramek et al., Arch.Biochem.Biophys. 171:14-26 (1975)) and has been shown to transfer the CoA moiety from acetyl-COA to a variety of substrates,
including isobutyrate (
Matthies et al., Appl Environ.Microbiol 58:1435-1439 (1992)), valerate and butanoate (
Vanderwinkel et al., Biochem.Biophys.Res.Commun. 33:902-908 (1968)). Similar enzymes exist in
Corynebacterium glutamicum ATCC 13032 (
Duncan et al., Appl.Environ.Microbiol 68:5186-5190 (2002)),
Clostridium acetobutylicum (
Cary et al., Appl.Environ.Microbiol 56:1576-1583 (1990);
Wiesenborn et al., Appl.Environ.Microbiol 55:323-329 (1989)), and
Clostridium saccharoperbutylacetonicum (
Kosaka. et al., Biosci.Biotechnol Biochem. 71:58-68 (2007)).
Gene |
GenBank Accession No. |
GI No. |
Organism |
cat1 |
P38946.1 |
729048 |
Clostridium kluyveri |
cat2 |
P38942.2 |
172046066 |
Clostridium kluyveri |
cat3 |
EDK35586.1 |
146349050 |
Clostridium kluyveri |
TVAG_395550 |
XP_001330176 |
123975034 |
Trichomonas vaginalis G3 |
Tb11.02.0290 |
XP_828352 |
71754875 |
Trypanosoma brucei |
gctA |
CAA57199.1 |
559392 |
Acidaminococcus fermentans |
gctB |
CAA57200.1 |
559393 |
Acidaminococcus fermentans |
gctA |
ACJ24333.1 |
212292816 |
Clostridium symbiosum |
gctB |
ACJ24326.1 |
212292808 |
Clostridium symbiosum |
atoA |
P76459.1 |
2492994 |
Escherichia coli K12 |
atoD |
P76458.1 |
2492990 |
Escherichia coli K12 |
actA |
YP_226809.1 |
62391407 |
Corynebacterium glutamicum |
cg0592 |
YP_224801.1 |
62389399 |
Corynebacterium glutamicum |
ctfA |
NP_149326.1 |
15004866 |
Clostridium acetobutylicum |
ctfb |
NP_49327.1 |
15004867 |
Clostridium acetobutylicum |
ctfA |
AAP42564.1 |
31075384 |
Clostridium saccharoperbutylacetonicum |
ctfB |
AAP42565.1 |
31075385 |
Clostridium saccharoperbutylacetonicum |
[0267] 3.1.2.a CoA hydrolase: 3-Oxo-3-phenylpropionyl-CoA can be hydrolyzed to its corresponding
acid by a CoA hydrolase or thioesterase in the EC class 3.1.2
(Step B of Figure 3). Exemplary CoA thioesters that hydrolyze aromatic substrates include benzoyl-CoA
hydrolase, 4-hydroxybenzoyl-CoA hydrolase (EC 3.1.2.23) and phenylacetyl-CoA hydrolase
(EC 3.1.2.25). The
Azoarcus evansii gene
orf1 encodes an enzyme with benzoyl-CoA hydrolase activity that participates in benzoate
metabolism (
Ismail, Arch.Microbiol 190:451-460 (2008)). This enzyme, when heterotogously expressed in
E. coli, demonstrated activity on a number of alternate substrates. Additional benzoyl-CoA
hydrolase enzymes were identified in benzonate degradation gene clusters of
Magnetospirillum magnetotacticum,
Jannaschia sp. CCS1 and
Sagittula stellata E-37 by sequence similarity (
Ismail, Arch.Microbiol 190.451-460 (2008)). The 4-hydroxybenzoyl-CoA hydrolase of
Pseudomonas sp. CBS3 exhibits activity on the alternate substrates benzoyl-CoA and p-methylbenzoyl-CoA,
and has heterologously expressed and characterized in
E. coli (
Song et al., Bioorg. Chem. 35:1-10 (2007)). Additional enzymes with aryl-CoA hydrolase activity include the palmitoyl-CoA
hydrolase of
Mycobacterium tuberculosis (
Wang et al., Chem.Biol. 14:543-551 (2007)) and the acyl-CoA hydrolase of
E. coli encoded by
entH (
Guo et al., Biochemistry 48:1712-1722 (2009)).
Gene |
GenBank Accession No. |
GI No. |
Organism |
orf1 |
AAN39365.1 |
23664428 |
Azoarcus evansii |
Magn03011230 |
ZP_00207794 |
46200680 |
Magnetospirillum magnetotacticum |
Jann_0674 |
YP_508616 |
89053165 |
Jannaschia sp. CCS1 |
SSE37-24444 |
ZP_01745221 |
126729407 |
Sagittula stellata |
EF569604.1:4745..5170 |
ABQ44580.1 |
146761194 |
Pseudomonas sp. CBS3 |
Rv0098 |
NP_214612.1 |
15607240 |
Mycobacterium tuberculosis |
entH |
AA.C73698.1 |
1786813 |
Escherichia coli |
[0268] Several additional CoA hydrolases with broad substrate ranges are suitable for hydrolyzing
benzoyl-CoA and/or p-methylbenzoyl-CoA. For example, the enzyme encoded by
acot12 from
Rattus norvegicus brain (
Robinson et al., Biochem.Biophys.Res.Commun. 71:959-965 (1976)) can react with butyryl-CoA, hexanoyl-CoA and malonyl-CoA. The human dicarboxylic
acid thioesterase, encoded by
acot8, exhibits activity on glutaryl-CoA, adipyl-CoA, suberyl-CoA, sebacyl-CoA, and dodecanedioyl-CoA
(
Westin et al., J.Biol.Che. 280:38125-38132 (2005)). The closest
E. coli homolog to this enzyme,
tesB, can also hydrolyze a range of CoA thiolesters (
Naggert et al., J Biol Chem 266:11044-11050 (1991)). A similar enzyme has also been characterized in the rat liver (
Deana R., Biochem Int 26:767-773 (1992)). Additional enzymes with hydrolase activity in
E.
coli include
ybgC, paaI, and ybdB (
Kuznetsova, et al., FEMS Microbiol Rev, 2005, 29(2):263-279;
Song et al., J Biol Chem, 2006, 281(16):11028-38).
Gene name |
GenBank Accession # |
GI# |
Organism |
acot12 |
NP_570103.1 |
18543355 |
Rattus norvegicus |
tesB |
NP_414986 |
16128437 |
Escherichia coli |
acot8 |
CAA15502 |
3191970 |
Homo sapiens |
acot8 |
NP_570112 |
51036669 |
Rattus norvegicus |
tes4 |
NP_415027 |
16128478 |
Escherichia coli |
ybgC |
NP_415264 |
16128711 |
Escherichia coli |
paaI |
NP_415914 |
16129357 |
Escherichia coli |
ybdB |
NP_415129 |
16128580 |
Escherichia coli |
[0269] 4.1.1a. Carboxy-lyase: Decarboxylase enzymes in the EC class 4.1.1 are required to
catalyze several transformations in Figures 1-3. Conversion of phenylpyruvate to phenylacetaldehyde
(Step B of Figure 1) is catalyzed by a keto-acid decarboxylase, Decarboxylation of
phenylacetate to toluene (Step D of Figure 1) is catalyzed by an enzyme with phenylacetate
decarboxylase, activity. A 3-oxoacid decarboxylase is required to decarboxylate benzoyl-acetate
to acetophenone (Step C of Figure 7). Decarboxylases (also known as carboxy lyases)
catalyze the loss of carbon dioxide from an organic compound or a cellular metabolite
possessing a carboxylic acid function. Decarboxylases are prevalent in nature and
can require either pyridoxal phosphate or pyruvate as a co-factor, although many require
no bound co-factors. Over 50 decarboxylase enzymes have been reported and characterized
by biochemical and/or analytical methods.
[0270] A phenylacetate decarboxylase is required to catalyze the decarboxylation of phenylacetate
to toluene (
Step D of Figure 1). Such an activity has not been detected in characterized decarboxylase enzymes to
date. An enzyme catalyzing a similar reaction is 4-hydroxyphenylacetate decarboxylase
(EC 4.1.1.83), which naturally decarboxylates 4-hydroxyphenylacetate to p-cresol.
Characterized 4-hydroxyphenylacetate decarboxylase enzymes from
Clostridium difficile and
Clostridium scatologenes are composed of two subunits and require activation by a specific activating enzyme
(
Yu et al., Biochemistry 45:9584-9592 (2006);
Andrei et al., Eur.J Biochem. 271:2225-2230 (2004)). These enzymes, encoded by
csdABC in
C. scatologenes and
hpdABC in
C. difficile, have been heterotogously expressed in
E. coli. Another suitable enzyme is the arylmalonate decarboxylase (EC 4.1.1.76) of
Enterobacter cloacae, which has activity on the structurally related 2-phenylpropionate (
Yatake et al., Appl. Microbiol Biotechnol. 78:793-799 (2008)). The sequence of this enzyme is available in the publication by Yatake
et al; however this enzyme has not been assigned a GenBank Accession number to date. A
related enzyme from
Bordetella bronchiseptica (98% amino acid identity) encoded by
AMDA_BORBR was recently crystallized (
Kuettner et al., J Mol.Biol. 377:386-394 (2008)).
Gene name |
GenBank Accession # |
GI# |
Organism |
csdA |
ABB05048.1 |
77863917 |
Clostridium scatologenes |
csdB |
ABB05046.1 |
77863915 |
Clostridium scatologenes |
csdC |
ABB05047.1 |
77863916 |
Clostridium scatologenes |
hpdA |
CAD6589L1.1 |
28300943 |
Clostridium difficile |
hpdB |
CAD65889.1 |
28300939 |
Clostridium difficile |
hpdC |
CAD65890.1 |
28300941 |
Clostridium difficile |
AMDA_BORBR |
Q05115.1 |
728844 |
Bordetella bronchiseptica |
[0271] The conversion of phenylpyruvate to phenylacetaldehyde (
Figure_
1, Step B) is catalyzed by an enzyme with phenylpyruvate decarboxylase activity. Several keto-acid
decarboxylase enzymes have demonstrated activity on phenylpyruvate including phenylpyruvate
decarboxylase (EC 4.1.1.43), pyruvate decarboxylase (EC 4.1.1.1), branched-chain alpha-ketoacid
decarboxylase (EC 4.1.1.72) and benzylformate decarboxylase (EC 4.1.1.7). An exemplary
phenylpyruvate decarboxylase is encoded by the
ipdC gene of
Azospirillum brasilense (
Spaepen et al., J Bacteriol. 189:7626-7633 (2007)). Phenylpyruvate is the favored substrate of this enzyme. The
Saccharomyces cerevisiae enzymes encoded by the genes
PDC1, PDC5, PDC6 and
ARO10 also exhibit phenylpyruvate decarboxylase activity (
Dickinson et al., J Biol.Chem. 278:8028-8034 (2003)). Other enzymes with phenylpyruvate decarboxylase activity include the pyruvate
dehydrogenase enzyme
Zygosaccharomyces bisporus (
Neuser et al., Biol. Chem. 381:349-353 (2000)), the branched chain 2-oxoacid decarboxylase enzymes
of Mycobacterium tuberculosis H37Rv and
Lactococcus lactis (Gocke et al., Adv.Synth.Catal. 349:1425-1435 (2007); Werther et al., J Biol.Chem. 283:5344-5354 (2008)), and the benzylformate decarboxylase enzyme of
Pseudomonas putida (
Siegert et al., Protein Eng Dees Sel 18:345-357 (2005)). Another suitable enzyme is the pyruvate decarboxylase from
Zymomonas mobilus, as this enzyme has a broad substrate range and has been a subject of directed engineering
studies to alter the substrate specificity (
Siegert et al., Protein Eng Des Sel 18:345-357 (2005)).
Protein |
GenBank ID |
GI Number |
Organism |
ipdC |
P51852.1 |
1706320 |
Azospirillum brasilense |
PDC1 |
P06169.7 |
30923172 |
Saccharomyces cerevisiae |
PDC5 |
P16467.4 |
1352225 |
Saccharomyces cerevisiae |
PDC6 |
P26263.3 |
118389 |
Saccharomyces cerevisiae |
ARO10 |
Q06408.1 |
50400299 |
Saccharomyces cerevisiae |
pdc1 |
CAB65554.1 |
6689662 |
Zygosaccharomyces bisporus |
Rv0853c |
NP_215368.1 |
15607993 |
Mycobacterium tuberculosis H37Rv |
kdcA |
AAS49166.1 |
44921617 |
Lactococcus lactis |
mdlC |
P20906.2 |
3915757 |
Pseudomonas putida |
pdc |
P06672.1 |
118391 |
Zymomonas mobilis |
[0272] The decarboxylation of benzoyl-acetate to acetophenone (
Step C of Figure 3) is catalyzed by an enzyme with benzoyl-acetate decarboxylase activity. An ATP-dependent
enzyme with this activity, acetophenone carboxylase, operates in the reverse (carboxylation)
direction during ethylbenzene degradation in
Aromatoleum aromaticum EbN1 (
Rabus et al., Arch.Microbiol 178:506-516 (2002)). This enzyme is composed of five subunits encoded by
apc1-5 and is ATP-dependent (AMP forming) in the direction of carboxylation. Similar enzymes
are found by sequence homology in
Azotobacter vinelandii and
Rubrobacter xylanophilus.
Gene name |
GenBank Accession # |
GI# |
Organism |
apc1 |
YP_158351.1 |
56476762 |
Aromatoleum aromaticum EbN1 |
apc2 |
YP_158350.1 |
56476761 |
Aromatoleum aromaticum EbN1 |
apc3 |
YP_158349.1 |
56476760 |
Aromatoleum aromaticum EbN1 |
apc4 |
YP_158348.1 |
56476759 |
Aromatoleum aromaticum EbN1 |
apc5 |
YP_158347.1 |
56476758 |
Aromateleum aromaticum EbN1 |
apc1 |
YP_002798345.1 |
226943272 |
Azotobacter vinelandii |
apc2 |
ACO77369.1 |
226718198 |
Azotobacter vinelandii |
apc3 |
YP_002798343.1 |
226943270 |
Azotobacter vinelandii |
apc4 |
YP_002798342.1 |
226943269 |
Azotobacter vinelandii |
apc1 |
YP_644617.1 |
108804680 |
Rubrobacter xylanophilus |
apc2 |
YP_644616.1 |
108804679 |
Rubrobacter xylanophilus |
apc3 |
YP_644615.1 |
108804678 |
Rubrobacter xylanophilus |
apc4 |
YP_644614.1 |
108804677 |
Rubrobacter xylanophilus |
apc5 |
YP_644613.1 |
108804676 |
Rubrobacter xylanophilus |
[0273] Alternatively, a 3-oxoacid decarboxylase can convert benzoyl-acetate to acetophenone.
An exemplary 3-oxoacid decarboxylase is acetoacetate decarboxylase (EC 4.1.1.4), which
naturally converts acetoacetate into acetone and CO
2. The enzyme from
Clostridium acetobutylicum, encoded by
adc, has a broad substrate range and has been shown to catalyze the desired decarboxylation
of benzoyl-acetate to acetophenone (
Rozzel et al., J.Am.Chem.Soc. 106:4937-4941 (1984);
Benner et al., J.Am.Chem.Soc. 103:993-994 (1981);
Autor et al., J Biol. Chem. 245:5214-5222 (1970)). A related acetoacetate decarboxylase has been characterized in
Clostridium beijerinckii (
Ravagnani et al., Mol.Microbiol 37:1172-1185 (2000)). The acetoacetate decarboxylase from
Bacillus polymyxa, characterized in cell-free extracts, also has a broad substrate specificity for
3-keto acids and can decarboxylate 3-oxopentanoate (
Matiasek et al., Curr.Microbiol 42:276-281 (2001)). The gene encoding this enzyme has not been identified to date and the genome sequence
of
B.
polymyxa is not yet available. Another
adc is found in
Clostridium saccharoperbutylacetonicum (
Kosaka, et al., Biosci.Biotechnol Biochem. 71-58-68 (2007)). Additional genes in other organisms, including
Clostridium botulinum and
Bacillus amyloliquefaciens FZB42, can be inferred by sequence homology.
Protein |
GenBank ID |
GI Number |
Organism |
adc |
NP_149328.1 |
15004868 |
Clostridium acetobutylicum |
adc |
AAP42566.1 |
31075386 |
Clostridium saccharoperbutylacetonicum |
adc |
YP_001310906.1 |
150018652 |
Clostridium beijerinckii |
CLL_A2135 |
YP_001886324.1 |
187933144 |
Clostridium botulinum |
RBAM_030030 |
YP_001422565.1 |
154687404 |
Bacillus amyloliquefaciens |
[0274] 4.1.99.a Decarbonylase: A decarbonylase enzyme is required to convert phenylacetaldehyde
to toluene (
Step E of Figure 1). Decarbonylase enzymes catalyze the final step of alkane biosynthesis in plants,
mammals, insects and bacteria (
Dennis et al., Arch. Biochem.Biophys. 287:268-275 (1991)). Non-oxidative decarbonylases transfom aldehydes into alkanes with the concurrent
release of CO, whereas oxidative 3decarboxylases are cytochrome P450 enzymes that
utilize NADPH and O
2 as cofactors and release CO
2, water and NADP
+. Exemplary decarbonylase enzymes include octadecanal decarbonylase (EC 4.1.99.5),
sterol desaturase and fatty aldehyde decarbonylase. A cobalt-porphyrin containing
decarbonylase was purified and characterized in the algae
Botryococcus braunii; however, no gene is associated with this activity to date (
Dennis et al., Proc.Natl.Acad.Sci.U.S.A 89:5306-5310 (1992)). A copper-containing decarbonylase from
Pisum sativum was also purified and characterized (
Schneider-Belhaddad et al., Arch.Biochem.Biophys. 377:341-349 (2000)). The
CER1 gene of Arabidopsis thaliana encodes a fatty acid decarbonylase involved in epicuticular
wax formation (
US 6,437,218). Additional fatty acid decarbonylases are found in
Medicago truncatula, Vitis vinifera and
Oryza sativa (
US Patent Application 2009/0061493).
Protein |
GenBank ID |
GI Nunber |
Organism |
CER1 |
NP_850932 |
145361948 |
Arabidopsis thaliana |
MtrDRAFT_AC153128g2v2 |
ABN07985 |
124359969 |
Medicago trunincatula |
VITISV_029045 |
CAN60676 |
147781102 |
Vitis vinifera |
OSJNBa0004N05.14 |
CAE03390.2 |
38345317 |
Oryza sativa |
[0275] Oxidative decarbonylase enzymes are encoded by the
CYP4G2v1 and
CYP4G1 gene products of
Musca domestica and
Drosophila melanogaster (
US Patent Application 2010/0136595). Additional enzymes with oxidative decarbonylase activity can be identified in other
organisms, for example
Mamestra brassicae, Helicoverpa zea and
Acyrthosiphon pisum, by sequence homology.
Protein |
GenBank ID |
GI Number |
Organism |
CYP4G2v1 |
ABV48808.1 |
157382740 |
Musca domestica |
CYP4G1 |
NP_525031.1 |
17933498 |
Drosophila melanogaster |
CYP4G25 |
BAD81026.1 |
56710314 |
Antheraea yamamai |
CYP4M6 |
AAM54722.1 |
21552585 |
Helicoverpa zea |
LOC100164072 |
XP_001944205.1 |
193650239 |
Acyrthosiphon pisum |
[0276] 4.1.99.b Lyase: The conversion of phenylalanine to benzene in
Figure 2 is catalyzed by an enzyme with phenylalanine benzene-lyase activity. The required
novel activity is similar to that of tyrosine phenol-lyase (EC 4.1.99.2), which reversibly
interconverts tyrosine and phenol, with concomitant release of pyruvate and ammonia.
The enzyme from
Pantoea agglomerans (formerly
Erwinia herbicola), encoded by
tutA, reacts with a range of substituted derivatives including dihydroxyphenylalanine
(
Foor et al., Appl.Environ.Microbiol 59:3070-3075 (1993)). This enzyme was heterologously expressed in
E. coli and utilized to synthesize dihydroxyphenylalanine from catechol, pyruvate and ammonia.
Additional tyrosine phenol-lyase enzymes include are found in
Citrobacter intermedius (
Nagasawa et al., Eur.J Biochem. 117:33-40 (1981)),
Citrobacter freundii (
Phillips et al., Biochim.Biophys.Acta 1647:167-172 (2003)) and
Symbiobacterium thermophilum (
Hirahara et al., Appl.Microbiol Biotechnol. 39:341-346 (1993)). The
Citrobacter freundii enzyme has been structurally characterized and residues involved in substrate binding
were identified (
Milic et al., J Biol.Chem. 283:29206-29214 (2008)). Phenylalanine is not a natural substrate of any characterized tyrosine phenol-lyase
enzyme; rather it has been shown to act as inhibitor of some enzymes. Directed evolution
or other protein engineering approaches well known in the art will likely be required
to gain this activity and improve performance.
Protein |
GenBank ID |
GI Number |
Organism |
tutA |
AAA66390.1 |
806897 |
Pantoea agglomerans |
tpl |
ABI75028.1 |
114451977 |
Citrobacter freundii |
tpl |
BAA00763.1 |
216675 |
Citrobacter intermedius |
tpl |
YP_076671.1 |
51893980 |
Symbiobacterium thermophilum |
[0278] 4.2.1.a Dehydratase:
Step E of Figure 3 employs a dehydratase (EC 4.1.2.-) to convert 1-phenylethanol to styrene. Exemplary
enzymes for catalyzing this reaction include fumarase (EC 4.2.1.2), citramalate hydratase
(EC 4.2.1.34) and dimethylmaleate hydratase (EC 4.2.1.85). Fumarase enzymes naturally
catalyze the reversible dehydration of malate to fumarate. Although the suitability
of 1-phenylethanol as a substrate for fumarase enzymes has not been described in the
literature to date, a wealth of structural information is available for this enzyme
and researchers have successfully engineered the enzyme to alter activity, inhibition
and localization (Weaver, 61:1395-1401 (2005)).
E. coli has three fumarases: FumA, FumB, and FumC that are regulated by growth conditions.
FumB is oxygen sensitive and only active under anaerobic conditions. FumA is active
under microanaerobic conditions, and FumC is the only enzyme active during aerobic
growth (Tseng et al., 183:461-467 (2001); Woods et al., 954:14-26 (1988);
Guest et al., J Gen Microbiol 131:2971-2984 (1985)). Additional enzymes are found in
Campylobacter jejuni (
Smith et al., Int.J Biochem.Cell Biol 31:961-975 (1999)),
Thermus therophilus (
Mizobata et al., Arch.Biochem.Biophys. 355:49-55 (1998)) and
Rattus norvegicus (Kobayashi et al., 89:1923-1931 (1981)). Similar enzymes with high sequence homology include
fum1 from
Arabidopsis thaliana and
fumC from
Corynebacterium glutamicum. The
mmcBC fumarase from
Pelotomaculum thermopropionicum is another class of fumarase with two subunits (Shimoyama et al., 270:207-213 (2007)).
Citramalate hydrolyase naturally dehydrate 2-methylmalate to mesaconate. This enzyme
has been studied in
Methanocaldococcus jannaschii in the context of the pyruvate pathway to 2-oxobutanoate, where it has been shown
to have a broad specificity (
Drevland et al., J Bacteriol. 189:4391-4400 (2007)). This enzyme activity was also detected in
Clostridium tetanomorphum, Morganella morganii, Citrobacter amalonaticus where it is thought to participate in glutamate degradation (
Kato and Asano, Arch.Microbiol 168:457-463 (1997)). The
M. jannaschii protein sequence does not bear significant homotogy to genes in these organisms.
Dimethylmaleate hydratase is a reversible Fe
2+-dependent and oxygen-sensitive enzyme in the aconitase family that hydrates dimethylmaeate
to form (2R,3S)-2,3-dimethylmalate. This enzyme is encoded by
dmdAB in
Eubacterium barkeri (Alhapel et al.,
supra;
Kollmann-Koch et al., Hoppe Selers.Z.Physiol Chem. 365:847-857 (1984)).
Protein |
GenBank ID |
GI Number |
Organism |
fumA |
NP_416129.1 |
16129570 |
Escherichia coli |
fumB |
NP_418546.1 |
16131948 |
Escherichia coli |
fumC |
NP_416128.1 |
16129569 |
Escherichia coli |
fumC |
069294 |
9789756 |
Campylobacter jejuni |
fumC |
P84127 |
75427690 |
Thermus thermohilus |
fumH |
P14408 |
120605 |
Rattus norvegicus |
fumI |
P93033 |
39931311 |
Arabidopsis thaliana |
fumC |
Q8NRN8 |
39911596 |
Corynebacterium glutamicum |
mmcB |
YP_001211906 |
147677691 |
Pelotomacuum thermopropionicum |
mmcC |
YP_001211907 |
147677692 |
Pelotomaculum thermopropionicum |
leuD |
Q58673.1 |
3122345 |
Methanocaldococcus jannaschii |
dmdA |
ABC88408 |
86278276 |
Eubacterium barkeri |
dmdB |
ABC88409.1 |
86278277 |
Eubacterium barkeri |
[0279] 6.2.1.a Acid-thiol ligase: The conversion of 3-oxo-3-phenylpropionyl-CoA to benzoyl-acetate
(Step B of Figure 3) can be catalyzed by a CoA synthetase or acid-thiol ligase in the EC class 6.2.1.
ATP-forming CoA synthetases catalyzing this exact transformation have not been characterized
to date, however, Several enzymes with broad substrate specificities have been described
in the literature. The ADP-forming acetyl-COA synthetase (ACD, EC 6.2.1.13) from
Archaeoglobus fulgidus, encoded by AF1211, was shown to operate on a variety of linear and branched-chain
substrates including isobutyrate, isopentanoate, and fumarate (
Musfeldt et al., J Bacteriol. 184:636-644 (2002)). A second reversible ACD in
Archaeoglobus fulgidus, encoded by
AF1983, was also shown to have a broad substrate range with high activity on aromatic compounds
phenylacetate and indoleacetate (Musfeldt et al.,
supra). The enzyme from
Haloarcula marismortui, annotates as a succinyl-CoA synthetase, accepts propionate, butyrate, and branched-chain
acids (isovalerate and isobutyrate) as substrates, and was shown to operate in the
forward and reverse directions (
Brasen et al., Arch.Microbiol 182:277-287 (2004)). The ACD encoded by
PAE3250 from hyperthermophilic crenarchaeon
Pyrobaculum aerophilum showed the broadest substrate range of all characterized ACDs, reacting with acetyl-CoA,
isobutyryl-CoA (preferred substrate) and phenylacetyl-CoA (
Brasen and Schonheit, Arch.Microbiol 182:277-287 (2004)). Directed evolution or engineering can be used to modify this enzyme to operate
at the physiological temperature of the host organism. The enzymes from
A.
fulgidus, H. marismortui and
P. aerophilum have all been cloned, functionally expressed, and characterized in
E. coli (
Brasen and Schonheit, Arch.Microbiol 182:277-287 (2004);
Musfeldt and Schonheit, J Bacteriol. 184:636-644 (2002)). An additional enzyme is encoded by
sucCD in
E. coli, which naturally catalyzes the formation of succinyl-CoA from succinate with the concomitant
consumption of one ATP, a reaction which is reversible
in vivo (
Buck et al., Biochemistry 24:6245-6252 (1985)). The acyl CoA ligase from
Pseudomonas putida has been demonstrated to work on several aliphatic substrates including acetic, propionic,
butyric, valeric, hexanoic, heptanoic, and octanoic acids and on aromatic compounds
such as phenylacetic and phenoxyacetic acids (
Fernandez-Valverde et al., Appl.Environ.Microbiol. 59:1149-1154 (1993)). A related enzyme, malonyl CoA synthetase (6.3.4.9) from
Rhizobium leguminosarum could convert several diacids, namely, ethyl-, propyl-, allyl-, isopropyl-, dimethyl-,
cyclopropyl-, cyclopropytmethytene-, cyclobutyl-, and benzyl-matonate into their corresponding
monothioesters (
Pohl et al., J.Am.Chem.Soc. 123:5822-5823 (2001)).
Gene |
GenBank Accession No. |
GI No. |
Organism |
AF1211 |
NP_070039.1 |
11498810 |
Archaeoglobus fulgidus |
AF1983 |
NP_070807.1 |
11499565 |
Archaeoglobus fulgidus |
scs |
YP_135572.1 |
55377722 |
Haloarcula marismortui |
Gene |
GenBank Accession No. |
GI No. GI |
Organism |
PAE3250 |
NP_560604.1 |
18313937 |
Pyrobaculum aerophilum str. |
|
|
|
IM2 |
sucC |
NP_415256.1 |
16128703 |
Escherichia coli |
sucD |
AAC73823.1 |
1786949 |
Escherichia coli |
paaF |
AAC24333.2 |
22711873 |
Pseudomonas putida |
matB |
AAC83455.1 |
3982573 |
Rhizobium leguminosarum |
EXAMPLE II
Pathways to 1,3-Butadiene from Muconate Isomers
[0280] This example shows pathway from muconate isomers to 1,3 -butadiene.
[0281] Figure 4 shows the conversion of muconate isomers to 1,3-butadiene by decarboxylase
enzymes. Cis,cis-muconate, cis,trans-muconate or trans,trans-muconate is first decarboxylated
to either cis-2,4-pentadienoate or trans-2,4-pentadienoate (Steps A, B, C and D of
Figure 4). 2,4-Pentadienoate is subsequently decarboxylated to form 1,3-butadiene
(Steps E, F of Figure 4).
[0283] It is further understood that decarboxylation of either isomer of 2,4-pentadienoate
will form 1,3-butadiene. Isomers of 2,4-pentadienoate can alternatively be formed
from starting materials other than muconate (e.g., introduction of second double bond
via dehydrogenation of pent-2-enoate or pent-4-enoate; removal of CoA from 2,4-pentadienoyl-CoA
via a hydrolase, synthetase, or transferase, dehydrogenation of 2,4-pentadienal or
2,4-pentadienol via an aldehyde or aldehyde/alcohol dehydrogenase, respectively).
Isomers of 2,4-pentadienoate can be interconverted by isomerase enzymes in the EC
class: 5.2.1.
[0284] Numerous decarboxylase enzymes have been characterized and shown to decarboxylate
structurally similar substrates to muconate or 2,4-pentadienoate isomer. Exemplary
enzymes include sorbic acid decarboxylase, aconitate (EC 4.1.1.16), 4-oxalocrotonate
decarboxylase (EC 4.1.1.77), cinnamate decarboxylase and ferulic acid decarboxylase,
These enzymes are applicable for use in the present invention to decarboxylate muconate
and/or 2,4-petitadienoate as shown in Figure 4.
[0285] One decarboxylase enzyme with closely related function is sorbic acid decarboxylase
which, converts sorbic acid to 1,3-pentadiene. Sorbic acid decarboxylation by Aspergillus
niger requires three genes: padA1, ohbA1, and sdrA (
Plumridge et al. Fung. Genet. Bio, 47:683-692 (2010). PadA1 is annotated as a phenyiacrylic acid decarboxylase, ohbA1 is a putative 4-hydroxybenzoic
acid decarboxylase, and sdrA is a sorbic acid decarboxylase regulator. Additional
species have also been shown to decarboxylate sorbic acid including several fungal
and yeast species (
Kinderlerler and Hatton, Food Addit Contam., 7(5).657-69 (1990);
Casas et al., Int J Food Micro., 94(1):93-96 (2004);
Pinches and Apps, Int. J. Food Microbiol. 116: 182-185 (2007)). For example, Aspergillus oryzae and Neosartorya fischeri have been shown to decarboxylate
sorbic acid and have close homologs to
padA1, ohbA1, and
sdrA.
Gene name |
GenBankID |
GI Number |
Organism |
padA1 |
XP_001390532.1. |
145235767 |
Aspergillus niger |
ohbA1 |
XP_001390534.1 |
145235771 |
Aspergillus niger |
sdrA |
XP_001390533.1 |
145235769 |
Aspergillus niger |
padA1 |
XP_001818651.1 |
169768362 |
Aspergillus oryzae |
ohbA1 |
XP_001818650.1 |
169768160 |
Aspergillus oryzae |
sdrA |
XP_001818649.1 |
169768358 |
Aspergillus oryzae |
padA1 |
XP_001261423.1 |
119482790 |
Neosartorya fischeri |
ohbA1 |
XP_001261424.1 |
119482792 |
Neosartorya fischeri |
sdrA |
XP_001261422.1 |
119482788 |
Neosartorya fischeri |
[0286] Aconitate decarboxylase is another useful enzyme for this invention. This enzyme
catalyzes the final step in itaconate biosynthesis in a strain of
Candida and also in the filamentous fungus
Aspergil
lus terreus. (
Bonnarme et al. J Bacteriol. 177:3573-3578 (1995);
Willke and Vorlop, Appl Microbiol. Biotechnol 56:289-295 (2001)) Aconitate decarboxylase has been purified and characterized from
Aspergillus terreus. (
Dwiarti et al., J. Biosci. Bioeng. 94(1): 29-33 (2002)) The gene and protein sequence for the cis-aconitic acid decarboxylase (CAD) enzyme
were reported previously (
EP 2017344 A1;
WO 2009/014437 A1), along with several close homologs listed in the table below.
Gene name |
GenBandID |
GI Number |
Organism |
CAD |
XP_001209273 |
115385453 |
Aspergillus terreus |
|
XP_001217495 |
115402837 |
Aspergillus terreus |
|
XP_001209946 |
115386810 |
Aspergillus terreus |
|
BAE66063 |
83775944 |
Aspergillus oryzae |
|
XP_001393934 |
145242722 |
Aspergillus niger |
|
XP_391316 |
46139251 |
Gibberella zeae |
|
XP_001389415 |
145230213 |
Aspergillus niger |
|
XP_001383451 |
126133853 |
Pichia stipitis |
|
YP_891060 |
118473159 |
Mycobacterium smegmatis |
|
NP_961187 |
41408351 |
Mycobacterium avium subsp. pratuberculosis |
|
YP_880968 |
118466464 |
Mycobacterium avium |
|
ZP_01648681 |
119882410 |
Salinispora arenicola |
[0287] 4-Oxalocronate decarboxylase catalyzes the decarboxylation of 4-oxalocrotonate to
2-oxopentanoate. This enzyme has been isolated from numerous organisms and characterized.
Genes encoding this enzyme include
dmpH and
dmpE in
Pseudomonas sp. (strain 600) (Shingler et al., 174:711-724 (1992)),
xyIII and
xyIII from
Pseudomonas putida (
Kato et al., Arch.Microbiol 168:457-463 (1997);
Stanley et al., Biochemistry 39:3514 (2000);
Lian et al., J.Am.Chem.Soc. 116:10403 -10411 (1994)) and
Reut_B5691 and
Reut_B5692 from
Ralstonia eutropha JMP134 (Hughes et al., 158:79-83 (1984)). The genes encoding the enzyme from
Pseudomonas sp. (strain 600) have been cloned and expressed in
E. coli (Shingler et al., 174:711-724 (1992)).
Gene name |
GenBankID |
GI Number |
Organism |
dmpH |
CAA43228.1 |
45685 |
Pseudomonas sp. CF600 |
dmpE |
CAA43225.1 |
45682 |
Pseudomonas sp. CF600 |
xylII |
YP_709328.1 |
111116444 |
Pseudomonas putida |
xylIII |
YP_709353.1 |
111116469 |
Pseudomonas putida |
Reut_B5691 |
YP_299880.1 |
73539513 |
Ralstonia eutropha JMP134 |
Reut_B5692 |
YP_299881.1 |
73539514 |
Ralstonia eutropha JMP134 |
[0288] Finally, a class of decarboxylases has been characterized that catalyze the conversion
of cinnamate (phenylacrylate) and substituted cinnamate derivatives to their corresponding
styrene derivatives. These enzymes are common in a variety of organisms and specific
genes encoding these enzymes that have been cloned and expressed in
E. coli include:
pad 1 from
Saccharomyces cerevisae (
Clausen et al., Gene 142.107-112 (1994)),
pdc from
Lactobacillus plantarum (Barthelmebs et al., 67:1063-1069 (2001);
Qi et al., Metab Eng 9:268-276 (2007);
Rodriguez et al., J.Agric.Food Chem. 56:3068-3072 (2008)),
pofk (
pad) from
Klebsielia oxytoca (
Uchiyama et al., Biosci.Biotechnol.Bioehem. 72:116-123 (2008);
Hashidoko et al., Biosci.Biotech.Biochem. 58:21,7-218 (1994)),
Pedicoccus pentosaceus (Barthelmebs et al., 67:1063-1069 (2001)), and
padC from
Bacillus subtily and
Bacillus pumilus (Shingler et al., 174-711-724 (1992)). A ferulic acid decarboxylase from
Pseudomonas fluorescens also has been purified and characterized (
Huang et al., J.Bacteriol. 176:5912-5918 (1994)). Enzymes in this class are stable and do not require either exogenous or internally
bound co-factors, thus making these enzymes ideally suitable for biotransformations
(
Sariaslani, Annu.RevMicrobiol. 61:51-69 (2007)).
Gene name |
GenBankID |
GI Number |
Organism |
pad1 |
BAG32372.1 |
188496949 |
Saccharomyces cerevisae |
pdc |
AAC45282.1 |
1762616 |
Lactobacillus plantrum |
pofK (pad) |
BAF65031.1 |
149941607 |
Klebsiella oxytoca |
padC |
AAC46254.1 |
2394282 |
Bacillus subtilis |
pad |
CAC16793.1 |
11322457 |
Pedicoccus pentosaceus |
pad |
CAC18719.1 |
11691810 |
Bacillus pumilus |
[0289] Each of the decarboxylases listed above a possible suitable enzyme for the desired
transformation shown in Figure 4. If the desired activity or productivity of the is
not observed in the desired conversions (e.g., muconate to 2,4-pentadienoate, 2,4-pentadienoate
to butadiene), the decarboxylase enzymes can be evolved using known protein engineering
methods to achieve the required, performance. Importantly, it was shown through the
use of chimeric enzymes that the C-terminal region of decarboxylases appears to be
responsible for substrate specificity (
Barthetmebs, L.; Divies, C.; Cavin, J.-F. 2001. Expression in Escherichia coli of
Native and Chimeric Phenolic Acid Decarboxylases with Modified Enzymatic Activities
and Method for Screening Recombinant E. coli Strains Expressing These Enzymes, Appl.
Environ. Microbiol. 67, 1063-1069.). Accordingly, directed, evolution experiments to broaden the specificity of decarboxylases
in to gain activity with muconate or 2,4-pentadienoate can be focused on the C-terminal
region, of these enzymes.
[0290] Some of the decarboxylases required to catalyze the transformations in
Figure 4 may exhibit higher activity on specific isomers of muconate or 2,4-pentadienoate.
Isomerase enzymes can be applied to convert less desirable isomers of muconate and
2,4-pentadienoate into more desirable for decarboxylation. Exemplary that catalyze
similar transformations and thus represent suitable enzymes for this invention include
maleate cis-trans isomerase (EC 5.2.1.1), maleytacetone cis-trans isomerase (EC 5.2.1.2)
and fatty acid cis-trans isomerase. Maleate cis-trans isomerase converts fumarate
to maleate, This enzyme is encoded by the
maiA gene from
Alcaligenes faecalis (
Hatakeyama, et al., 1997, Gene Cloning and Characterization of Maleate cis-trans Isomerase
from Alcaligenes faccalis, Biochem. Biophys. Research Comm. 239, 74-79) or
Serratia marcescens (
Hatakeyama et al., Biosci. Biotechnol. Biochem. 64:1477-1485 (2000)). Similar genes that can be identified by sequence homology include those from
Geobacillus stearothermophilus, Ralstonia pickettii 12D, and
Ralstonia eutropha H16. Additional maleate cis-trans isomerase enzymes are encoded by the enzymes whose
amino acid sequences are provided as sequence ID's 1 through 4 in ref (
Mukouyama et al., US Patent 6,133,014). Maleytacetone
cis,trans-isomerase catalyzes the conversion of 4-maleyl-acetoacetate to 4-fumaryl-acetyacetate,
a cis to trans conversion. This enzyme is encoded by
maiA in
Pseudomonas aeruiginosa (
Fernandez-Canon et al., J Biol.Chem. 273:329-337 (1998))) and
Vibirio cholera (
Seltzer, J Biol.Chem. 248:215-222 (1973)). A similar enzyme was identified by sequence homology in
E. coli O157. The
cti gene product catalyzes the conversion of
cis- unsaturated fatty acids (UFA) to
trans- UFA. The enzyme has been characterized in
P. putida (
Junker et at., J Bacteriol. 181:5693-5700 (1999)). Similar enzymes are found in
Shewanella sp. MR-4 and
Vibrio cholerae.
Gene name |
GenBankID |
GI Number |
Organism |
maiA |
BAA23002.1 |
2575787 |
Alcaligenes faecalis |
maiA |
BAA96747.1 |
8570038 |
Serratia marcescens |
maiA |
BAA77296 |
4760466 |
Geobacillus stearothermophilus |
Rpic12DDRAFT_0600 |
ZP_02009633 |
153888491 |
Ralstonia piekettii 12D |
maiA |
YP_725437 |
113866948 |
Ralstonia eutropha H16 |
maiA |
NP_250697 |
15597203 |
Pseudomonas aeruginosa |
maiA |
NP_230991 |
15641359 |
Vibrio cholerae |
maiA |
EDU73766 |
189355347 |
Escherichia coli O157 |
cti |
AAD41252 |
5257178 |
Pseudomonas putida |
cti |
YP_732637 |
113968844 |
Shewanella sp. MR-4 |
cti |
ZP_04395785 |
229506276 |
Vibrio cholerae |
EXAMPLE III
Exemplary Pathway for Producing (2-Hydroxy-3-methyl-4-oxobutoxy)phosphonate
[0291] This example describes an exemplary pathway for producing the terephthalic acid (PTA)
precursor (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate (2H3M4OP).
[0292] The precursor to the p-toluate and PTA pathways is 2H3M4OP. This chemical can be
derived from central metabotites glyceraldehyde-3-phosphate (G3P) and pyruvate in
three enzymatic steps as shown in Figure 5. The first two steps are native to E. coli
and other organisms that utilize the methytlerythritol phosphate (non-mevalonate)
pathway for isoprenoid biosynthesis. Pyruvate and G3P are first condensed to form
1-deoxy-D-xylulose 5-phosphate (DXP) by DXP synthase. Subsequent reduction and rearrangement
of the carbon backbone is catalyzed by DXP reductoisomerase. Finally, a novel diol
dehydratase transforms 2-C-methyl-D-erythritol-4-phosphate to the p-toluate precursor
2H3M4OP.
[0293] A. 1- Deoxyxylulose-5 -phosphate (DXP) synthase. Pyruvate and G3P are condensed to form DXP by DXP synthase (EC 2.2.1.7). This enzyme
catalyzes the first step in the non-mevalonate pathway of isoprenoid biosynthesis.
The enzyme requires thiamine diphosphate as a cofactor, and also requires reduced
FAD, although there is no net redox change. A crystal structure of the E. coli enzyme
is available (
Xiang et al., J. Biol. Chem. 282:2676-2682 (2007)). Other enzymes have been cloned and characterized in M. tuberculosis (
Bailey et al., Glycobiology 12:813-820 (2002) and Agrobacterium tumefaciens (
Lee et al., J. Biotechnol. 128:555-566 (2007)). DXP synthase enzymes from
B.subtilis and
Synechocystis sp. PCC 6803 were cloned into
E. coli (
Harker and Bramley, FEBS Lett. 448:115-119 (1999)).
Gene |
GenBank Accession No. |
GI No. |
Organism |
dxs |
AAC73523.1 |
1786622 |
Escherichia coli |
dxs |
P0A554.1 |
61222979 |
M. tuberculosis |
dxs11 |
AAP56243.1 |
37903541 |
Agrobacterium tumefaciens |
dxs |
P54523.1 |
1731052 |
Bacillus subtilis |
sll1945 |
BAA17089.1 |
1652165 |
Synechocytis sp. PCC 6803 |
[0295] C. 2-C-Methyl-D-erythritol-4-phosphate, dehydratase. A diol dehydratase is required. to convert 2-C-methyl-D-erythritol-4-phosphate into
the
p-toluate precursor (
Altmiller and Wagner, Arch. Biochem. Biophys. 138:160-170 (1970)). Although this transformation has not been demonstrated experimentally, several
enzymes catalyze similar transformations including dihydroxy-acid dehydratase (EC
4.2.1.9), propanediol dehydratase (EC 4.2.1.28), glycerol dehydratase (EC 4.2.1.30)
and myo-inositose dehydratase (EC 4.2.1.44).
[0296] Diol dehydratase or propanediol. dehydratase enzymes (EC 4.2.1.28) capable of converting
the secondary diol 2,3-butanediol to 2-butanone are excellent candidates for this
transformation. Adenosylcobatamin-dependent diol dehydratases contain alpha, beta
and gamma subunits, which are all required for enzyme function. Exemplary gene candidates
are found in
Klebsielli pneumoniae (
Tobimatsu et al., Biosci. Bioteclinol. Biochem. 62:1774-1777 (1998);
Toraya et at.,. Biochem. Biophys. Res. Commun. 69:475-480 (1976)),
Salmonella typhimurium. (
Bobik et al., J. Bacteriol. 179:6633-6639 (1997)),
Klebsiella oxytoca (
Tobimatsu et al., J. Biol. Chem. 270:7142-7148 (1995)) and
Lactobacillus collinoides (
Sauvageot et al., FEMS Microbiol. Lett. 209:69-74 (2002)). Methods for isolating diol dehydratase gene candidates in other organisms are
well known in the art (see, for example,
U.S. Patent No. 5,686,276).
Gene |
GenBank Accession No. |
GI No. |
Organism |
pddA |
BAA08099.1 |
868006 |
Klebsiella oxytoca |
pddB |
BAA08100.1 |
868007 |
Klebsiella oxytoca |
pddC |
BAA08101.1 |
868008 |
Klebsiella oxytoca |
pduC |
AAB84102.1 |
2587029 |
Salmonella typhimurium |
pduD |
AAB84103.1 |
2587030 |
Salmonella typhimurium |
pduE |
AAB94104.1 |
2587031 |
Salmonella typhimurium |
pduC |
CAC82541.1 |
18857678 |
Lactobacullus collinoides |
pduD |
CAC82542.1 |
18857679 |
Lactobacullus collinoides |
pduE |
CAD01091.1 |
18857680 |
Lactobacullus collinoides |
pddA |
AAC98384.1 |
4063702 |
Klebsiella pneumoniae |
pddB |
AAC98385.1 |
4063703 |
Klebsiella pneumoniae |
pddC |
AAC98386.1 |
4063704 |
Klebsiella pneumoniae |
[0297] Enzymes in the glycerol dehydratase family (EC 4.2.1.30) can also be used to dehydrate
2-C-methyl-D-erythritol-4-phosphate. Exemplary gene candidates encoded by
gldABC and
dhaB123 in
Klebsiella pneumoniae (
WO 2008/137403) and (
Toraya et al., Biochem. Biophys. Res. Commun. 69:475-480 (1976)),
dhaBCE in
Clostridium pasteuranum (
Macis et al., FEMS Microbial Lett. 164:21-28 (1998)) and
dhaBCE in
Citrobacter freundii (
Seyfried et al., J. Bacteriol. 178:5793-5796 (1996)). Variants of the B12-dependent diol dehydratase from
K.
pneumoniae with 80- to 336-fold enhanced activity were recently engineered by introducing mutations
in two residues of the beta subunit (
Qi et al., J. Biotechnol. 144:43-50 (2009)). Diol dehydratase enzymes with reduced inactivation kinetics were developed by
DuPont using error-prone PCR (
WO 2004/056963).
Gene |
GenBank Accession No. |
GI No. |
Organism |
gldA |
AAB96343.1 |
1778022 |
Klebsiella pneumoniae |
gldB |
AAB96344.1 |
1778023 |
Klebsiella pneumoniae |
gldC |
AAB96345.1 |
1778024 |
Klebsiella pneumoniae |
dhaB1 |
ABR78884.1 |
150956854 |
Klebsiella pneumoniae |
dhaB2 |
ABR78883.1 |
150956853 |
Klebsiella pneumoniae |
dhaB3 |
ABP78882.1 |
150956852 |
Klebsiella pneumoniae |
dhaB |
AAC27922.1. |
3360389 |
Clostridium pasteuranum |
dhaC |
AAC27923.1 |
3360390 |
Clostridium pasteuranum |
dhaE |
AAC27924.1 |
3360391 |
Clostridium pasteuranum |
dhaB |
P45514.1 |
1169287 |
Citrobacter freundii |
dhaC |
AAB48851.1 |
1229154 |
Citrobacter freundii |
dhaE |
AAB48852.1 |
1229155 |
Citrobacter freundii |
[0298] If a B12-dependent diol dehydratase is utilized, heterologous expression of the corresponding
reactivating factor is recommended. B12-dependent diol dehydratases are subject to
mechanism-based suicide activation by substrates and some downstream products. Inactivation,
caused by a. tight association with inactive cobalamin, can be partially overcome
by diol dehydratase reactivating factors in an AT
P-dependent process. Regeneration of the B12 cofactor requires an additional ATP. Diol
dehydratase regenerating factors are two-subunit proteins. Exemplary candidates are
found in
Klebsiella oxytoca (
Mori et al., J. Biol. Chem. 272:32034-32041 (1997)),
Salmonella typhimurium (
Bobik et al., J. Bacteriol. 179:6633-6639 (1997);
Chen et al., J. Bacteriol. 176:5474-5482 (1994)),
Lactobacillus collinoides (
Sauvageot et al., FEMS Microbiol. Lett. 209:69-74 (2002)), and
Klebsiella pneumonia (
WO 2008/137403).
Gene |
GenBank Accession No. |
GI No. |
Organism |
ddrA |
AAC15871.1 |
3115376 |
Klebsiella oxytoca |
ddrB |
AAC15872.1 |
3115377 |
Klebsiella oxytoca |
pduG |
AAL20947.1 |
16420573 |
Salmonella typhimurium |
pduH |
AAL20948.1 |
16420574 |
Salmonella typhimurium |
pduG |
YP_002236779 |
206579698 |
Klebsiella pneumonia |
pduH |
YP_002236778 |
206579863 |
Klebsiella pneumonia |
pduG |
CAD01092 |
29335724 |
Lactobacillus collinoides |
pduH |
CAD01093 |
29335725 |
Lactobacillus collinoides |
[0299] B12-independent diol dehydratase enzymes utilize S-adenosylmethionine (SAM) as a
cofactor, function under strictly anaerobic conditions, and require activation by
a specific activating enzyme (
Frey et al., Chem. Rev. 103:2129-2148 (2003)). The glycerol dehydrogenase and corresponding activating factor of
Clostridium butyricum, encoded by
dhaB1 and
dhaB2, have been well-characterized (
O'Brien et al., Biochemistry 43:4635-4645 (2004);
Raynaud et al., Proc. Natl. Acad. Sci USA 100:5010-5015 (2003)). This enzyme was recently employed in a 1,3-propanediol overproducing strain of
E. coli and was able to achieve very high titers of product (
Tang et al., Appl. Environ. Microbiol. 75:1628-1634 (2009)). An additional B12-independent diol dehydratase enzyme and activating factor from
Roseburia inulinivorans was shown to catalyze the conversion of 2,3-butanediol to 2-butanone (
US publication 2009/09155870).
Gene |
GenBank Accession No. |
GI No. |
Organism |
dhaB1 |
AAM54728.1 |
27461255 |
Clostridium butyricum |
dhaB2 |
AAM54729.1 |
27461256 |
Clostridium butyricum |
rdhtA |
ABC25539.1 |
83596382 |
Roseburia inulinivorans |
rdhtB |
ABC25540.1 |
83596383 |
Roseburia inulinivorans |
[0300] Dihydroxy-acid dehydratase (DHAD, EC 4.2.1.9) is a B12-independent enzyme participating
in branched-chain amino acid biosynthesis, In its native role, it converts 2,3-dihydroxy-3-methylvaterate
to 2-keto-3-methyl-valerate, a precursor of isoleucine. In valine biosynthesis, the
enzyme catalyzes the dehydration of 2,3-dihydroxy-isovalerate to 2-oxoisovalerate.
The DHAD from
Sulfolobus solfataricus has a broad substrate range, and activity of a recombinant enzyme expressed in
E.
coli was demonstrated on a variety of aldonic acids (
Kim and Lee, J. Biochem. 139:591-596 (2006)). The
S. solfataricus enzyme is tolerant of oxygen unlike many diol dehydratase enzymes. The
E. coli enzyme, encoded by
ilvD, is sensitive to oxygen, which inactivates its iron-sulfur cluster (
Flint et al., J. Biol. Chem. 268:14732-14742 (1993)), Similar enzymes have been characterized in
Neurospora crassa (
Altmiller and Wagner, Arch. Biochem. Biophys. 138:160-170 (1970)) and
Salmonella typhimurium (
Armstrong et al., Biochim. Biophys. Acta 498:282-293 (1977)).
Gene |
GenBank Accession No. |
GI No. |
Organism |
ilvD |
NP_344419.1 |
15899814 |
Sulfolobus solfataricus |
ilvD |
AAT48208.1 |
48994964 |
Escherichia coli |
ilvD |
NP_462795.1 |
16767180 |
Salmonella typhimurium |
ilvD |
XP_958280.1 |
85090149 |
Neurospora crassa |
[0301] The diol dehydratase myo-inosose-2-dehydratase (EC 4.2.1.44) is another exemplary
candidate. Myo-inosose is a. six-membered ring containing adjacent alcohol groups.
A purified enzyme encoding myo-inosose-2-dehydratase functionality has been studied
in
Klebsiella aerogenes in the context of myo-inositol degradation (
Berman and Magasanik, J. Biol. Chem. 241:800-806 (1966)), but has not been associated with a gene to date, The myo-inosose-2-dehydratase
of Sinorhizobium fredii was cloned and functionally expressed in
E.
coli (
Yoshida et al., Biosci. Biotechnol. Biochem. 70:2957-2964 (2006)). A similar enzyme from
B. subtilis, encoded by
iolE, has also been studied (
Yoshida et al., Microbiology 150-571-580 (2004)).
Gene |
GenBank Accession No. |
GI No. |
Organism |
iolE |
P42416.1 |
1176989 |
Bacillus subtilis |
iolE |
AAX24114.1 |
60549621 |
Sinorhizobium fredii |
EXAMPLE IV
Exemplary Pathway for Synthesis of p-Toluate from (2-Hydroxy-3-methyl-4-oxobutoxy)phosphonate
by Shikimate Pathway Enzymes
[0302] This example describes exemplary pathways for synthesis of
p-toluate using shikimate pathway enzymes.
[0303] The chemical structure of
p-toluate closely resembles
p-hydroxybenzoate, a precursor of the electron carrier ubiquinone. 4-Hydroxybenzoate
is synthesized from central metabolic precursors by enzymes in the shikimate pathway,
found in bacteria, plants and fungi. The shikimate pathway is comprised of seven enzymatic
steps that transform D-erythrose-4-phosphate (E4P) and phosphoenolpyruvate (PEP) to
chorismate. Pathway enzymes include 2-dehydro-3-deoxyphosphoheptonate (DAHP) synthase,
dehydroquinate (DHQ) synthase, DHQ dehydratase, shikimate dehydrogenase, shikimate
kinase, 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase and chorismate synthase.
In the first step of the pathway, erythrose-4-phosphate and phosphoenolpyruvate are
joined by DAHP synthase to form 3-deoxy-D-arabino-heptulosonate-7-phosphate. This
compound is then dephosphorylated, dehydrated and reduced to form shikimate. Shikimate
is converted to chorismate by the actions of three enzymes: shikimate kinase, 3-phosphoshikimate-2-carboxyvinyltransferase
and chorismate synthase. Subsequent conversion of chorismate to 4-hydroxybenzoate
is catalyzed by chorismate lyase.
[0304] The synthesis of p-toluate proceeds in an analogous manner as shown in. Figure 6.
The pathways originates with PEP and 2H3M4OP, a compound analogous to E4P with a methyl
group in place of the 3-hydroxyl group of E4P. The hydroxyl group of E4P does not
directly participate in the chemistry of the shikimate pathway reactions, so the methyl-substituted.
2H3M4OP precursor is expected to react as an alternate substrate. Directed or adaptive
evolution can be used to improve preference for 2H3M4OP and downstream derivatives
as substrates. Such methods are well-known in the art.
[0305] Strain engineering strategies for improving the efficiency of flux through shikimate
pathway enzymes are also applicable here. The availability of the pathway precursor
PEP can be increased by altering glucose transport systems (
Yi et al., Biotechnol. Prog. 19:1450-1459 (2003)). 4-Hydroxybenzoate-overproducing strains were engineered to improve flux through
the shikimate pathway by means of overexpression of a feedback-insensitive isozyme
of 3-deoxy-D-arabinoheptulosonic acid-7-phosphate synthase (
Barker and Frost, Biotechnol. Bioeng. 76:376-390 (2001)). Additionally, expression levels of shikimate pathway enzymes and chorismate lyase
were enhanced. Similar strategies can be employed in a strain for overproducing p-toluate.
[0306] A. 2-Dehydro-3-deoxyphosphoheptonate synthase (EC 2.5.1.54). The condensation of
erythrose-4-phosphate and phosphoenolpyruvate is catalyzed by 2-dehydro-3-deoxyphosphoheptonate
(DAMP) synthase (EC 2.5.1.54). Three isozymes of this enzyme are encoded in the E.
coli genome by aroG, aroF and aroH and are subject to feedback inhibition by phenylalanine,
tyrosine and tryptophan, respectively. In wild-type cells grown on minimal medium,
the aroG, aroF and aroH gene products contributed 80%, 20% and 1% of DAHP synthase
activity, respectively (
Hudson and Davidson, J. Mol. Biol. 180:1023-1051 (1984)). Two residues of AroG were found to relieve inhibition by phenylalanine (
Kikuchi et al., Appl. Environ. Microbiol. 63:761-762 (1997)). The feedback inhibition of AroF by tyrosine was removed by a single base-pair
change (
Weaver and Herrmann, J. Bacteriol. 172:6581-6584 (1990)). The tyrosine-insensitive DAHP synthase was overexpressed in a 4-bydroxybenzoate-overproducing
strain of E. coli (
Barker and Frost, Biotechnol. Bioeng. 76:376-390 (2001)). The aroG gene product was shown to accept a variety of alternate 4-and 5-carbon
length substrates (
Sheflyan et al., J. Am. Chem. Soc. 120(43):11027-11032 (1998);
Williamson et al., Bioorg. Med. Chem. Lett. 15:2339-2342 (2005)), The enzyme reacts efficiently with (3S)-2-deoxyerythose-4-phosphate, a substrate
analogous to erythrose-4-phosphate but lacking the alcohol at the 2-position (Williamson
et al., supra 2005). Enzymes from Helicobacter pylori and Pyrococcus furiosus also
accept this alternate substrate (
Schofield et al., Biochemistry 44:11950-11962 (2005));
Webby et al., Biochem. J. 390:223-230 2005)) and have been expressed in E. coli. An evolved variant of DAHP synthase, differing
from the wild type E. coli AroG enzyme by 7 amino acids, was shown to exhibit a 60-fold
improvement in Kcat/KM (
Ran and Frost, J. Am. Chem. Soc. 129:6130-6139 (2007)).
Gene |
GenBank Accession No. |
GI No. |
Organism |
aroG |
AAC73841.1 |
1786969 |
Escherichia coli |
aroF |
AAC75650.1 |
1788953 |
Escherichia coli |
aroH |
AAC74774.1 |
1787996 |
Escherichia coli |
aroF |
Q9ZMU5 |
81555637 |
Helicobacter pylori |
PF1690 |
NP_579419.1 |
18978062 |
Pyrococcus furiosus |
[0308] C. 3-Dehydroquinate dehydratase (EC 4.2.1.10). 3-Dehydroquinate dehydratase, also termed 3-
dehydroquinase (DHQase), naturally catalyzes the dehydration of 3-dehydroquinate to 3-dehyd
roshikimate, analogous to step C in the
p-toluate pathway of Figure 2. DHQase enzymes can be divided into two classes based
on mechanism, stereochemistry and sequence homology (
Gourley et al., Nat. Struct. Biol. 6.521-525. (1999)). Generally the type 1 enzymes are involved in biosynthesis, while the type 2 enzymes
operate in the reverse (degradative) direction. Type 1 enzymes from
E.
coli (
Kinghorn et al., Gene 14:73-80. 1981)),
Salmonella typhi (Kinghorn et al.,
supra 1981;
Servos et al., J. Gen. Microbiol. 137:147-152 (1991)) and
B. subtilis (
Warburg et al., Gene 32:57-66 1984)) have been cloned and characterized. Exemplary type II 3-dehydroquinate dehydratase
enzymes are found in
Mycobacterium tuberculosis, Streptomyces coelicolor (
Evans et al., FEBS LEtt. 530:24-30 (2002)) and
Helicobacter pylori (
Lee et al., Proteins 51:616-7 (2003)).
Gene |
GenBank Accession No. |
GI No. |
Organism |
aroD |
AAC74763.1 |
1787984 |
Escherichia coli |
aroD |
P24670.2 |
17433709 |
Salmonella typhi |
aroC |
NP_390189.1 |
16079365 |
Bacillus subtilis |
aroD |
P0A4Z6.2 |
61219243 |
Mycobacterium tuberculosis |
aroQ |
P15474.3 |
8039781 |
Streptomyces coelicolor |
aroQ |
Q48255.2 |
2492957 |
Helicobacter pylori |
[0309] D. Shikimate dehydrogenase (EC 1.1.1.25). Shikimate dehydrogenase catalyzes the NAD(P)H
dependent reduction of 3-dehydroshikimate to shikimate, analogous to Step D of Figure 2. The
E.
coli genome encodes two shikimate dehydrogenase paralogs with different cofactor specificities.
The enzyme encoded by
aroE is NADPH specific, whereas the
ydiB gene product is a quinate/shikimate dehydrogenase which can utilize NADH (preferred)
or NADPH as a cofactor (
Michel et al., J. Biol. Chem. 278:19463-19472 (2003)). NADPH-dependent enzymes from
Mycobacterium tuberculosis (
Zhang et al., J. Biochem. Mol. Biol. 38:624-631 (2005)),
Haemophilus influenzae (
Ye et al., J Bacteriol 185:4144-4151 (2003)) and
Helicobacter pylori (
Han et al., FEBS J. 273:4682-4692 (2006)) have been functionally expressed in
E. coli.
Gene |
GenBank Accession No. |
GI No. |
Organism |
aroE |
AAC76306.1 |
1789675 |
Escherichia coli |
ydiB |
AAC74762.1 |
1787983 |
Escherichia coli |
aroE |
NP_217068.1 |
15609689 |
Mycobacterium tuberculosis |
aroE |
P43876.1 |
1168510 |
Haemophilus influenzae |
aroE |
AAW22052.1 |
56684731 |
Helicobacter pylori |
[0310] E. Shikimate kinase (EC 2.7.1.71). Shikimate kinase catalyzes the AT
P-dependent phosphorylalion of the 3-hydroxyl group of shikimate analogous to Step
E of Figure 2. Two shikimate kinase enzymes are encoded by
aroK (SK1) and
aroL (SK2) in
E.
coli (
DeFeyter and Pittard, J. Bacteriol. 165:331-333 (1986);
Lobner-Olesen and Marinus, J. Bacteriol. 174:525-529 (1992)). The Km of SK2, encoded by
aroL, is 100-fold lower than that of SK1, indicating that this enzyme is responsible for
aromatic biosynthesis (DeFeyter et al.,
supra 1986). Additional shikimate kinase enzymes from
Mycobacterium tuberculosis (
Gu et al., J. Mol. Biol. 319:779-789 (2002));
Oliveira et al., Protein Expr. Purif. 22-430-435 (2001)(doi: 10.1006/prep.2001.1457, doi;S1046-5928(01)91457-3, pii),
Helicobacter pylori (
Cheng et al., J. Bacteriol. 187:8156-8163 (2005)) and
Erwinia chrysanthemi (
Krell et al., Protein Sci. 10:1137-1149 (2001)) have been cloned in
E.
coli.
Gene |
GenBank Accession No. |
GI No. |
Organism |
aroK |
YP_026215.2 |
90111581 |
Escherichia coli |
aroL |
NP_414922.1 |
16128373 |
Escherichia coli |
aroK |
CAB06199.1 |
1781063 |
Mycobacterium tuberculosis |
aroK |
NP_206956.1 |
15644786 |
Helicobacter pylori |
SK |
CAA32883.1 |
42966 |
Erwinia chrysanthemi |
[0314] B-F. Multifunctional AROM protein. In most bacteria, the enzymes of the shikimate pathway are encoded by separate polypeptides.
In microbial eukaryotes, five enzymatic functions are catalyzed by a polyfunctional
protein encoded by a pentafunctional supergene (
Campbell et al., Int. J. Parasitol. 34:5-13 (2004)). The multifunctional AROM protein complex catalyzes reactions analogous to reactions
B-F of Figure 2. The AROM protein complex has been characterized in fungi
including Aspergillus nidulans, Neurospora crassa, Saccharomyces cerevisiae and
Pneumocystis carinii (
Banerji et al., J. Gen. Microbiol. 139:2901-2914 (1993);
Charles et al., Nucleic Acids Res. 14:2201-2213 (1986);
Coggins et al., Methods Enzymol. 142:325-341 (1987);
Duncan, K., Biochem. J. 246:375-386 (1987)). Several components of AROM have been shown to function independently as individual
polypeptides. For example, dehydroquinate synthase (DHQS) forms the amino-terminal
domain of AROM, and can function independently when cloned into
E. coli (
Moore et al., Biochem. J. 301 (Pt 1):297-304 (1994)). Several crystal structures of AROM components from
Aspergillus nidulans provide insight into the catalytic mechanism (
Carpenter et al., Nature 394:299-302 (1998)).
Gene |
GenBank Accession No. |
GI No. |
Organism |
AROM |
P07547.3 |
238054389 |
Aspergillus nidulans |
AROM |
P08566.1 |
114166 |
Saccharomyces cerevisiae |
AROM |
P07547.3 |
238054389 |
Aspergillus nidulans |
AROM |
Q12659.1 |
2492977 |
Pneumocystis carinii |
EXAMPLE V
Exemplary Pathway for Enzymatic Transformation of p-Toluate to Terephthalic Acid
[0315] This example describes exemplary pathways for conversion of
p-toluate to terephthalic acid (PTA).
[0316] P-toluate can be further transformed to PTA by oxidation of the methyl group to an
acid in three enzymatic steps as shown in Figure 3. The pathway is comprised of a
p-toluate methyl-monooxygenase reductase, a 4-carboxybenzyl alcohol dehydrogenase and
a 4-carboxybenzyl aldehyde dehydrogenase. In the first step,
p-toluate methyl-monooxyngenase oxidizes
p-toluate to 4-carboxybenzyl alcohol in the presence of O2. The
Comamonas testosteroni enzyme (
tsaBM), which also reacts with 4-toluene sulfonate as a substrate, has been purified and
characterized (
Locher et al., J. Bacteriol. 173:3741-3748 (1991)). 4-Carboxybenzyl alcohol is subsequently converted to an aldehyde by 4-carboxybenzyl
alcohol dehydrogenase (
tsaC). The aldehyde to acid transformation is catalyzed by 4-carboxybenzaldehyde dehydrogenase
(
tsaD). Enzymes catalyzing these reactions are found
in Comamonas testosteroni T-2, an organism capable of utilizing
p-toluate as the sole source of carbon and energy (
Junker et al., J. Bacteriol. 179:919-927 (1997)). Additional genes to transform
p-toluate to PTA can be found by sequence homology, in particular to proteobacteria
in the genera
Burkholderia, Alcaligenes, Pseudomonas, Shingomonas and
Comamonas (
US Patent No. 6,187,569 and
US publication 2003/0170836). Genbank identifiers associated with the
Comamonas testosteroni enzymes are listed below.
Gene |
GenBank Accession No. |
GI No. |
Organism |
tsaB |
AAC44805.1 |
1790868 |
Comamonas testosteroni |
tsaM |
AAC44804.1 |
1790867 |
Comamonas testosteroni |
tsaC |
AAC44807.1 |
1790870 |
Comamonas testosteroni |
tsaD |
AAC44808.1 |
1790871 |
Comamonas testosteroni |
EXAMPLE VI
Synthesis of benzoate from 2H4OP
[0317] This example shows the synthesis of benzoate from 2H4OP by shikimate pathway enzymes
(Figure 9) and an exemplary pathway for producing the benzoate pathway precursor 2H4OP
(Figure, 8).
[0318] Like p-toluate, the chemical structure of benzoate resembles p-hydroxybenzoate, a
product of the shikimate pathway described above in Example IV. In this Example shikimate
pathway enzymes are utilized to synthesize benzoate from the pathway precursor (2-hydroxy-4-oxobutoxy)phosphonate
(2H4OP) by the pathway shown in Figure 9. The reactivity of shikimate pathway enzymes
on 2H4OP and the alternate substrates used in benzoate formation are optionally optimized
by directed or adaptive evolution to improve preference for 2H4OP and downstream derivatives
as substrates. Such methods are well-known in the art and described herein.
[0319] An exemplary and novel pathway for synthesizing the benzoate pathway precursor (2-hydroxy-4-oxobutoxy)phosphonate
(2H4OP) is shown in Figure 8. In this pathway, 2H4OP is derived from the central metabolite
erythrose-4-phosphate in two enzymatic steps. In the first step, a diol dehydratase
transforms crythrose-4-phosphate to (2,4-dioxobutoxy)phosphonate (24DBP). The 2-keto
group of 24DBP is subsequently reduced to the alcohol of 2H4OP by an oxidoreductase
with 24DBP reductase activity. Exemplary enzymes for Steps A and B of Figure 8 are
presented below.
[0320] A. Erythrose-4-phosphate dehydratase: Diol dehydratase enzymes in the EC class 4.2.1
are used to convert erythrose-4-phosphate into 24DBP (Figure 4, Step A). Although
enzymes catalyzing this transformation have not been indicated, several enzymes catalyze
similar transformations including dihydroxy-acid dehydratase (EC 4.2.1.9), propanediol
dehydratase (EC 4.2.1.28), glycerol dehydratase (EC 4.2.1.30) and myo-inositose dehydratase
(EC 4.2.1.44). Exemplary diol dehydratase enzymes are described above in Example III.
[0321] Diol dehydratase or propanediol dehydratase enzymes (EC 4.2.1.28) capable of converting
the secondary diol 2,3-butanediol to 2-butanone can be used for this transformation.
Adenosylcobalamin- or B12-dependent diol dehydratases contain alpha, beta and gamma
subunits, all of which are used for enzyme function. Exemplary genes are found in
Klebsiella pneumoniae (
Tobimatsu et al., Biosci. Biotechnol. Biochem. 62:1774-1777 (1998);
Toraya et al.,. Biochem. Biophys. Res. Commun. 69:475-480 (1976)),
Salmonella typhimurium (
Bobik et al., J. Bacteriol. 179:6633-6639 (1997)),
Klebsiella oxytoca (
Tobimatsu et al., J. Biol. Chem. 270:7142-7148 (1995)) and
Lactobacillus collinoides (
Sauvageot et al., FEMS Microbiol. Lett. 209:69-74 (2002)). Methods for isolating diol dehydratase genes in other organisms are well known
in the art as exemplified in
U.S. Patent No. 5,686,276, which is incorporated herein by reference in its entirety.
Gene |
GenBank Accession No. |
GI No. |
Organism |
pddA |
BAA08099.1 |
868006 |
Klebsiella oxytoca |
pddB |
BAA08100.1 |
868007 |
Klebsiella oxytoca |
pddC |
BAA08101.1 |
868008 |
Klebsiella oxytoca |
pduC |
AAB84102.1 |
2587029 |
Salmonella typhimurium |
pduD |
AAB84103.1 |
2587030 |
Salmonella typhimurium |
pduE |
AAB84104.1 |
2587031 |
Salmonella typhimurium |
pduC |
CAC82541.1 |
18857678 |
Lactobacullus collinoides |
pduD |
CAC82542.1 |
18857679 |
Lactobacullus collinoides |
pduE |
CAD01091.1 |
18857680 |
Lactobacullus collinoides |
pddA |
AAC98384.1 |
4063702 |
Klebsiella pneumoniae |
pddB |
AAC98385.1 |
4063703 |
Klebsiella pneumoniae |
pddC |
AAC98386.1 |
4063704 |
Klebsiella pneumoniae |
[0322] Enzymes in the glycerol dehydratase family (EC 4.2.1.30) can also be used to dehydrate
erythrose-4-phosphate. Exemplary genes include gldABC and dhaB123 in Klebsiella
pneumoniae (
WO 2008/137403) and (
Toraya et al., Biochem. Biophys. Res. Commun. 69:475-480 (1976)),
dhaBCE in
Clostridium pasteuranum (
Macis et al., FEMS Microbiol Lett. 164:21-28 (1998)) and dhaBCE in
Citrobacter freundii (
Seyfried et al., J. Bacteriol. 178:5793-5796 (1996)). Variants of the B12-dependent diol dehydratase from K.
pneumoniae with 80- to 336-fold enhanced activity were recently engineered by introducing mutations
in two residues of the beta subunit (
Qi et al., J. Biotechnol. 144:43-50 (2009)). Diol dehydratase enzymes with reduced inactivation kinetics were developed by
DuPont using error-prone PCR (
WO 2004/056963).
Gene |
GenBank Accession No. |
GI No. |
Organism |
gldA |
AAB96343.1 |
1778022 |
Klebsiella pneumoniae |
gldB |
AAB96344.1 |
1778023 |
Klebsiella pneumoniae |
gldC |
AAB96345.1 |
1778024 |
Klebsiella pneumoniae |
dhaB1 |
ABR78884.1 |
150956854 |
Klebsiella pneumoniae |
dhaB2 |
ABR78883.1 |
150956853 |
Klebsiella pneumoniae |
dhaB3 |
ABR78882.1 |
150956852 |
Klebsiella pneumoniae |
dhaB |
AAC27922.1 |
3360389 |
Clostridium pasteuranum |
dhaC |
AAC27923.1 |
3360390 |
Clostridium pasteuranum |
dhaE |
AAC27924.1 |
3360391 |
Clostridium pasteuranum |
dhaB |
P45514.1 |
1169287 |
Citrobacter freundii |
dhaC |
AAB48851.1 |
1229154 |
Citrobacter freundii |
dhaE |
AAB48852.1 |
1229155 |
Citrobacter freundii |
[0323] If a B12-dependent diol dehydratase is utilized, heterologous expression of the corresponding
reactivating factor is useful. B12-dependent diol dehydratases are subject to mechanism-based
suicide activation by substrates and some downstream products. Inactivation, caused
by a tight association with inactive cobalamin, can be partially overcome by diol
dehydratase deactivating factors in an ATP-dependent process. Regeneration of the
B12 cofactor is AT
P-dependent. Diol dehydratase regenerating factors are two-subunit proteins. Exemplary
genes are found
in Klebsiella oxytoca (
Mori et al., J. Biol. Chem. 272:32034-32041 (1997)),
Salmonella typhimurium (
Bobik et al., J. Bacteriol. 179.6633-6639 (1997);
Chen et al., J. Bacteriol. 176:5474-5482 (1994)),
Lactobacillus collinoides (
Sauvageot et al., FEMS Microbiol. Lett. 209:69-74 (2002)), and
Klebsiella pneumonia (
WO 2008/137403).
Gene |
GenBank Accession No. |
GI No. |
Organism |
ddrA |
AAC15871.1 |
3115376 |
Klebsiella oxytoca |
ddrB |
AAC15872.1 |
3115377 |
Klebsiella oxytoca |
pduG |
AAL20947.1 |
16420573 |
Salmonella typhimurium |
pduH |
AAL20948.1 |
16420574 |
Salmonella typhimurium |
pduG |
YP_002236779 |
206579698 |
Klebsiella pneumonia |
pduH |
YP_002236778 |
206579863 |
Klebsiella pneumonia |
pduG |
CAD01092 |
29335724 |
Lactobacillus collinoides |
pduH |
CAD01093 |
29335725 |
Lactobacillus collinoides |
[0324] B12-independent diol dehydratase enzymes utilize S-adenosylmethionine (SAM) as a
cofactor, function under strictly anaerobic conditions, and require activation by
a specific activating enzyme (
Frey et al., Chem. Rev. 103.2129-2148 (2003)). The glycerol dehydrogenase and corresponding activating factor of
Clostridium butyricum, encoded by
dhaB1 and
dhaB2, have been well-characterized (
O'Brien et al., Biochemistry, 43:4635-4645 (2004);
Raynaud et al., Proc. Natl. Acad. Sci USA 100:5010-5015 (2003)). This enzyme was recently employed in a 1,3-propanediol overproducing strain of
E.
coli and was able to achieve very high titers of product (
Tang et al., Appl. Environ. Microbiol. 75:1628-1634 (2009)). An additional B12-independent diol dehydratase enzyme and activating factor from
Roseburia inulinivorans was shown to catalyze the conversion of 2,3-butanediol to 2-butanone (
US publication 2009/09155870).
Gene |
GenBank Accession No. |
GI No. |
Organism |
dhaB1 |
AAM54728.1 |
27461255 |
Clostridium butyricum |
dhaB2 |
AAM54729.1 |
27461256 |
Clostridium butyricum |
rdhtA |
ABC25539.1 |
83596382 |
Roseburia inulinivorans |
rdhtB |
ABC25540.1 |
83596383 |
Roseburia inulinivorans |
[0325] Dihydroxy-acid dehydratase (DHAD, EC 4.2.1.9) is a B12-tndependent enzyme participating
in branched-chain amino acid biosynthesis. In its native role, it converts 2,3-dihydroxy-3-methylvalerate
to 2-keto-3-methyl-valerate, a precursor of isoleucine. In valine biosynthesis, the
enzyme catalyzes the dehydration of 2,3-dihydroxy-isovaterate to 2-oxoisovalerate.
The DHAD from
Sulfolobus solfataricus has a broad substrate range, and activity of a recombinant enzyme expressed in
E.
coli was demonstrated on a variety of aldonic acids (
Kim and Lee, J. Biochem. 139:591-596 (2006)). The
S. Solfataricus enzyme is tolerant of oxygen unlike many diol dehydratase enzymes. The
E. coli enzyme, encoded by
ilvD, is sensitive to oxygen, which inactivates its iron-sulfur cluster (
Flint et al., J. Biol. Chem. 268:14732-14742 (1993)). Similar enzymes have been characterized
in Neurospora crassa (
Altmiller and Wagner, Arch. Riochem. Biophys. 138:160-170 (1970)) and
Salmonella typhimurium (
Armstrong et al., Biochim. Biophys. Acta 498:282-293 (1977)).
Gene |
GenBank Accession No. |
GI No. |
Organism |
ilvD |
NP_344419.1 |
15899814 |
Sulfolobus solfataricus |
ilvD |
AAT48208.1 |
48994964 |
Escherichia coli |
ilvD |
NP_462795.1 |
16767180 |
Salmonella typhimurium |
ilvD |
XP_958280.1 |
85090149 |
Neurospora crassa |
[0326] The diol dehydratase myo-inosose-2-dehydratase (EC 4.2.1.44) is another exemplary
enzyme. Myo-inosose is a six-membered ring containing adjacent alcohol groups. A purified
enzyme encoding myo-inosose-2-dehydratase functionality has been studied in
Klebsiella aerogenes in the context of myo-inositol degradation (
Berman and Magasanik, J. Biol. Chem. 241:800-806 (1966)), but has not been associated with a gene to date. The myo-inosose-2-dehydratase
of
Sinorhizobium fredii was cloned and functionally expressed in
E.
coli (
Yoshida et al., Biosci. Biotechnol. Biochem. 70:2957-2964 (2006)). A similar enzyme from
B. subtilis, encoded by
iolE, has also been studied (
Yoshida et al., Microbiology 150:571-580(2004)).
Gene |
GenBank Accession No. |
GI No. |
organism |
iolE |
P42416.1 |
1176989 |
Bacillus subtilis |
iolE |
AAX24114.1 |
60549621 |
Sinorhizobium fredii |
[0327] B. (2,4-Dioxobutoxy)phosphonate reductase: An enzyme with (2,4-dioxobutoxy)phosphonate
reductase activity is used to convert 24DBP to 2H4OP. Although this compound is not
a knowns substrate of alcohol dehydrogenase enzymes, several enzymes in the EC class
1.1.1 catalyze similar reactions. Exemplary enzymes that reduce ketones of phosphorylated
substrates include glycerol-3-phosphate dehydrogenases (EC 1.1.1.8), 3-phosphoglycerate
dehydrogenase (EC 1.1.1.95) and erythronate-4-phosphate dehydrogenase (EC 1.1.1.290).
Glycerol-3-phosphate dehydrogenase catalyzes the NAD(P)H-dependent reduction of dihydroxyacetone-phosphate
to glycerol-3-phosphate. This enzyme is encoded by
gpsA of
E.
coli (
Kito et al., J Biol.Chem. 244:3316-3323 (1969)). 3-Phosphoglycerate dehydrogenase catalyzes the first step of serine biosynthesis
in several organisms including
E. coli, where it is encoded by the gene
serA (
Tobey et al., J Biol.CheM. 261:12179-12183 (1986)). Erythronate-4-phosphate dehydrogenase (EC 1.1.1.290) catalyzes the reversible
reduction of 2-oxo-3-hydroxy-4-phosphobutanoate to erythronate-4-phosphate, This enzyme,
encoded by
pdxB of
E. coli, normally operates in the reverse direction in the context of pyridoxal 5'-phosphate
biosynthesis (
Schoenlein et al., J Bacteriol, 171:6084-6092 (1989)). A similar enzyme encoded by
pdxB in
Pseudomonas aeruginosa has been characterized and heterologously expressed in
E. coli (
Ha et al., Acta Crystallogr.Sect.F.Struct.Biol.Cryst.Commun. 62:139-141 (2006)).
Gene |
GenBank Accession No. |
GI No. |
Organism |
gpsA |
AAC76632.1 |
1790037 |
Escherichia coli |
serA |
AAC75950.1 |
1789279 |
Escherichia coli |
pdxB |
AAC75380.1 |
1788660 |
Escherichia coli |
pdxB |
AAG04764.1 |
9947319 |
Pseudomonas aeruginosa |
[0328] A wide variety of alcohol dehydrogenase enzymes catalyze the reduction of a ketone
to an alcohol functional group. Two such enzymes from
E. coli are encoded by malate dehydrogenase (
mdh) and lactate dehydrogenase (
ldhA). The lactate dehydrogenase from
Ralstonia eutropha has been shown to demonstrate high activities on 2-ketoacids of various chain lengths
including lactate, 2-oxobutyrate, 2-oxopentanoate and 2-oxoglutarate (
Steinbuchel et al., Eur.J.Biochem. 130:329-334 (1983)). Conversion of alpha-ketoadipate into alpha-hydroxyadipate can be catalyzed by
2-ketoadipate reductase, an enzyme reported to be found in rat and in human placenta
(
Suda et al., Arch.Biochem.Biophys. 176:610-620 (1976);
Suda et al., Biochem.Biophys.Res.Commun. 77:586-591 (1977)). An additional oxidoreductase is the mitochondrial 3-hydroxybutyrate dehydrogenase
(
bdh) from the human heart which has been cloned and characterized (
Marks et al., J.Biol.Chem. 267:15459-15463 (1992)). Alcohol dehydrogenase enzymes of
C. beijerinckii (
Ismaiel etal., J.Bacteriol. 175:5097-5105 (1993)) and
T. brockii (
Lamed et al., Biochem.J. 195:183-190 (1981);
Peretz et al., Biochemistry. 28:6549-6555 (1989)) convert acetone to isopropanol. Methyl ethyl ketone reductase, or alternatively,
2-butanol dehydrogenase, catalyzes the reduction of MEK to form 2-butanol. Exemplary
enzymes can be found in
Rhodococcus ruber (
Kosjek et al., Biotechnol Bioeng. 86:55-62 (2004)) and
Pyrococcus furiosus (
van der et al., Eur.J.Biochem. 268:3062-3068 (2001)).
Gene |
GenBank Accession No. |
GI No. |
Organism |
mdh |
AAC76268.1 |
1789632 |
Escherichia coli |
ldhA |
NP_415898.1 |
16129341 |
Escherichia coli |
ldh |
YP_725.182.1 |
113866693 |
Ralstonia eutropha |
bdh |
AAA58352.1 |
177198 |
Homo sapiens |
adh |
AAA23199.2 |
60592974 |
C/ostridium beijerinckii NRRL B593 |
adh |
P14941.1 |
113443 |
Thermoanaerobacter brockii HTD4 |
adhA |
AAC25556 |
3288810 |
Pyrococcus furiosus |
sadh |
CAD36475 |
21615553 |
Rhodococcus ruber |
[0329] A number of organisms can catalyze the reduction of 4-hydroxy-2-butanone to 1,3-butanediol,
including those belonging to the genus
Bacillus, Brevibacterium, Candida, and
Klebsiella among others, as described by Matsuyama et al. ((1995)). A mutated
Rhodococcus pbenylacetaldehyde reductase (Sar268) and a
Leifonia alcohol dehydrogenases have also been shown to catalyze this transformation at high
yields (
Itoh et al., Appl.Microbiol Biotechnol. 75:1249-1256 (2007)).
[0330] Homoserine dehydrogenase (EC 1.1.1.13) catalyzes the NAD(P)H-dependent reduction
of aspartate semialdehyde to homoserine. In many organisms, including
E. coli, homoserine dehydrogenase is a bifunctional enzyme that also catalyzes the AT
P-dependent conversion of aspartate to aspartyl-4-phosphate (
Starnes et al., Biochemistry 11:677-687 (1972)). The functional domains are catalytically independent and connected by a linker
region (
Sibilli et al., J Biol.Chem. 256:10228-10230 (1981)) and both domains are subject to allosteric inhibition by threonine. The homoserine
dehydrogenase domain of the
E. coli enzyme, encoded by
thrA, was separated from the aspartate kinase domain, characterized, and found to exhibit
high catalytic activity and reduced inhibition by threonine (
James et al., Biochemistry 41:3720-3725(2002)). This can be applied to other bifunctional threonine kinases including, for example,
hom1 of
Lactobacillus plantarum (Cahyanto et al., Microobiology 152:105-112 (2006)) and
Arabidopsis thaliana. The monofunctional homoserine dehydrogenases encoded by
hom6 in
S. cerevisiae (
Jacques et al., Biochim.Biphoys.Acta 1544:28-41 (2001)) and
hom2 in
Lactobacillus plantarum (
Cahyanto et al., Microbiology 152:105-112 (2006)) have been functionally expressed and characterized in
E. coli.
Gene |
GenBank Accession No. |
GI No. |
Organism |
thrA |
AAC73113.1 |
1786183 |
Escherichia coli K12 |
akthr2 |
081852 |
75100442 |
Arabidopsis thaliana |
hom6 |
CAA89671 |
1015880 |
Saccharomyces cerevisiae |
hom1 |
CAD64819 |
28271914 |
Lactobacillus plantarum |
hom2 |
CAD63186 |
28270285 |
Lactobacillus plantarum |
EXAMPLE VII
Pathways to Benzene and Toluene
[0331] This Example shows pathways from benzoate and benzoyl-CoA to benzene, and
p-toluate and
p-methylbenzoyl-CoA to toluene.
[0332] Pathways for enzymatic production of benzene from benzoate or benzoyl-CoA are shoen
in Figure 10. Benzoate and benzoyl-CoA are naturally occurring metabolic intermediates
common to diverse aromatic degradation pathways. Benzoate can also be produced by
the alternate shikimate pathway route described herein. Several routes from benzoate
and/or benzoyl-CoA to benzene are shown in Figure 10. First, benzene can be produced
directly from benzoate via decarboxylation (Figure 10, path E). Alternately, the acid
moiety can be reduced to an aldehyde either directly by a benzoic acid reductase (Figure
10, path B) or indirectly via benzoyl-CoA and/or (benzoyloxy)phosphonate intermediates
(Figure 10, paths A and D, path B, path H and G, or path A, F, and G). Benzaldehyde
can be subsequently decarbonylated to form, benzene (Figure 10, path C).
[0333] Similar pathways for enzymatic production of toluene from
p-toluate and/or
p-methylbenzoyl-CoA are shown in Figure 11.
p-Toluate can be produced by the alternate shikimate pathway as described herein. Routes
for converting
p-toluate (also known as
p-methylbenzoate) and/or
p-methylbenzoyl-CoA to toluene are analogous to the transformation of benzoate or benzoyl-CoA
to benzene, described above. Enzymes for catalyzing the transformations shown in Figures
10 and 11 are categorized by EC number and described further below.
Label |
Function |
Fig-Path |
1.2.1.b |
Oxidoreductase (acyl-CoA to aldehyde) |
6/7-D |
1.2.1.d. |
Oxidoreductase (phosphorylating/dephosphorylating) |
6/7-G |
1.2.1.e |
Oxidoreductase (acid to aldehyde) |
6/7-B |
2.3.1.a |
Acyltransferase (transferring phosphate group to CoA; phosphotransacylase) |
6/7-F |
2.7.2.a |
Phosphotransferase, carboxyl group acceptor (kinase) |
6/7-H |
2.8.3.a |
Coenzyme-A transferase |
6/7-A |
3.1.2.a |
Thiolester hydrolase (CoA specific) |
6/7-A |
4.1.1.a |
Carboxy-lyase |
6/7-E |
4.1.99.a |
Decarbonylase |
6/7-C |
6.2.1.a. |
Acid-thiol ligase |
6/7-A |
[0334] 1.2.1.b Oxidoreductase (acyl-CoA. to aldehyde): An enzyme with benzoyl-CoA reductase
activity is used to convert benzoyl-CoA into benzaldehyde (Path D of Figure 10). Similarly,
the reduction of
p-methylbenzoyl-CoA to
p-methylbenzaldehyde (also called
p-tolualdehyde) is catalyzed by an enzyme with
p-methylbenzoyl-CoA reductase activity (Path D of Figure 11). Although enzymes with
these activities have not been characterized, an enzyme catalyzing a similar reaction
is cinnamoyl-CoA reductase (EC 1.2.1.44). This enzyme catalyzes the NAD(P)H-dependent
reduction of cinnamoyl-CoA and substituted aromatic derivatives such as coumaroyl-CoA
and feruloyl-CoA. The enzyme has been characterized in organisms including
Arabidopsis thaliana (Lauvergeat et al., Phytochemistry 57:1187-1195 (2001)), Triticum aestivum (Ma, J Exp.Bot. 58:2011-2021 (2007)) and
Panicum virgatum (Escamilla-Trevino et al., New Phytol. 185:143-155 (2010)). The enzymes from
A. thaliana and
P. virgatum where characterized and heterologously expressed in
E.
coli.
Gene |
GenBank Accession No. |
GI No. |
Organism |
TACCR1 |
ABE01883.1 |
90902167 |
Triticum aestivum |
AtCCR1 |
AAU45042.1 |
52355804 |
Arabidopsis thaliana |
AtCCR2 |
AAG53687.1 |
12407990 |
Arabidopsis thaliana |
PvCCR1 |
ACZ74580.1 |
270315096 |
Panicum virgatum |
PvCCR2 |
ACZ74585.1 |
270315106 |
Panicum virgatum |
[0335] Several other well-characterized acyl-CoA. reductases reduce an acyl-CoA to its corresponding
aldehyde. Exemplary enzymes include fatty acyl-CoA reductase (EC 1.2.1.50), succinyl-CoA
reductase (EC 1.2.1.76), acetyl-CoA reductase (EC 1.2.1.10) and butyryl-CoA reductase.
Exemplary fatty acyl-CoA reductases enzymes are encoded by
acr1 of
Acinetobacter calcoaceticus (Reiser et al., 179-2969-2975 (1997)) and
Acinetobacter sp. M-1 (
Ishige et al., Appl.Environ.Microbiol. 68:1192-1195 (2002)). Enzymes with succinyl-CoA reductase activity are encoded by
sucD of
Clostridium kluyveri (
Sohling et al., J Bacteriol. 178:871-880 (1996a); Sohling et al., 178:871-80 (1996)) and
sucD of
P.
gingivalis (
Takahashi et al., J.Bacteriol, 182:4704-4710 (2000)). Additional succinyl-CoA reductase enzymes participate in the 3-hydroxypropionate/4-hydroxybutyrate
cycle of thermophilic archaea such as
Metallosphaera sedula (
Berg et al., Science. 318:1782-1786 (2007)) and
Thermproteus neutrophilus (
Ramos-Vera et al., J Bacteriol. 191:4286-4297 (2009)), The
M.
sedula CoA reductase, encoded by
Msed_0709, is NADPH-dependent and also has malonyl-CoA, reductase activity. The
T. neutrophilus enzyme is active with both NADPH and NADH. The acylating acetaldehyde dehydrogenase
in
Pseudomonas sp, encoded by
bphG, has been demonstrated to oxidize and acrylate acetaldehyde, propionaldehyde, butyraldehyde,
isobutyraldehyde and formaldehyde to their corresponding CoA-esters (Powtowski et
al., 17.5:377-385 (1993)). In addition to reducing acetyl-CoA to ethanol, the enzyme
encoded by
adhE in
Leuconostoc mesenteroides has been shown to oxidize the branched, chain compound isoburtyraldehyde to isobutyryl-CoA
(
Koo et al., Biotechnol Lett. 27:505-510 (2005)). Butyraldehyde dehydrogenase catalyzes a similar reaction, conversion of butytyl-CoA
to butyraldehyde, in solventogenic organisms such as
Clostridium, saccharoperbutylacetonicum (
Kosaka et al., Biosci.Biotechnol Biochem. 71:58-68 (2007)). The acyl-CoA reductase encoded by
ald in
Clostridium beijerinckii has been indicated to reduce acetyl-CoA and butyryl-CoA to their corresponding aldehydes
(
Toth et al., Appl Environ.Microbiol 65:4973-4980 (1999)). This enzyme exhibits high sequence homology to the CoA-dependent acetaldehyde
dehydrogenase enzymes of
Salmonella typhimurium and
E.
coli encoded by
eutE (
Toth et al., Appl Environ.Microbiol 65:4973-4980 (1999)).
Gene |
GenBank Accession No. |
GI No. |
Organism |
acr1 |
YP-047869.1 |
50086359 |
Acinetobacter calcoaceticus |
acr1 |
AAC45217 |
1684886 |
Acinetobacter baylyi |
acr1 |
BAB85476.1 |
18857901 |
Acinetobacter sp. Strain M-1 |
MSED_0709 |
YP_001190808.1 |
146303492 |
Metallosphaera sedula |
Tneu_0421 |
ACB39369.1 |
170934108 |
Thermoproteus neutrophilus |
sucD |
P38947.1 |
172046062 |
Clostridium kluyveri |
sucD |
NP_904963.1 |
34540484 |
Porphyromonas gingivalis |
bphG |
BAA03892.1 |
425213 |
Pseudomonas sp |
adhE |
AAV66076.1 |
55818563 |
Leuconostoc mesenteroides |
bld |
AAP42563.1 |
31075383 |
Clostridium saccharoperbutylacetonicum |
ald |
AAT66436 |
9473535 |
Clostridium beijerinckii |
eutE |
AAA80209 |
687645 |
Salmonella typhimurium |
eutE |
P77445 |
2498347 |
Escherichia coli |
[0336] An additional CoA reductase enzyme is malonyl-CoA reductase which transforms malonyl-CoA
to malonic semialdehyde. Malonyl-CoA reductase is a key enzyme in autotrophic carbon
fixation via the 3-hydroxypropionate cycle in thermoacidophilic archael bacteria (Berg
et al., 318:1782-1786 (2007); Thauer, 318:1732-1733 (2007)). The enzyme utilizes NADPH
as a cofactor and has been characterized in
Metallosphaera and
Sulfolobus spp (Alber et al., 188:8551-8559 (2006); Hugler et al., 184:2404-2410 (2002)). The enzyme
is encoded by
Msed_0709 in
Metallosphaera sedula (Alber et al., 188:8551-8559 (2006); Berg et al., 318:1782-1786 (2007)). A gene encoding
a malonyl-CoA reductase from
Sulfolobus tokodaii was cloned and heterotogously expressed in
E. coli (Alber et al., 188:8551-8559 (2006)). This enzyme has also been indicated to catalyze
the conversion of methylmatonyl-CoA to its corresponding aldehyde (
WO/2007/141.208). Both malonyl-CoA reductase enzyme candidates have high sequence similarity to aspartate-semialdehyde
dehydrogenase, an enzyme catalyzing the reduction and concurrent dephosphorylation
of aspartyl-4-phosphate to aspartate semialdehyde. Additional genes can be found by
sequence homology to proteins in other organisms including
Sulfolobus solfataricus and
Sulfolobus acidocaldarius.
Gene |
GenBank Accession No. |
GI No. |
Organism |
MSED_0709 |
YP_001190808.1 |
146303492 |
Metallosphaera sedula |
mcr |
NP_378167.1 |
15922498 |
Sulfolobus tokodaii |
asd-2 |
NP_343563.1 |
15898958 |
Sulfolobus solfataricus |
Saci_2370 |
YP_250941.1 |
70608071 |
Sulfolobus acidocaldarius |
[0337] 1,2.1.d Oxidoreductase (phosphorylating/dephosphorylating) (10/11 G): The reductions
of (benzoyloxy)phosphonate to benzaldehyde (Path G of Figure 10) and (
p-methylbenzoyloxy) phosphonate to
p-methylbenzaldehyde (Path G of Figure 11) are catalyzed by enzymes with phosphonate
reductase activities. Although enzymes catalyzing these conversions have not been
identified to date, similar transformations catalyzed by glyceraldehyde-3-phosphate
dehydrogenase (EC 1.2.1.12), aspartate-semialdehyde dehydrogenase (EC 1.2.1.11) acetylglutamylphosphate
reductase (EC 1.2.1.38) and glatamate-5-semialdehyde dehydrogenase (EC 1.2.1.) are
well documented. Aspartate semialdehyde dehydrogenase (ASD, EC 1.2.1.11) catalyzes
the NADPH-dependent reduction of 4-aspartyl phosphate to aspartate-4-semialdehyde.
ASD participates in amino acid biosynthesis and recently has been studied as an antimicrobial
target (
Hadfield et al., Biochemistry 40:14475-14483 (2001)). The
E. coli ASD structure has been solved (
Hadfield et al., J Mol. Biol. 289:991-1002 (1999)) and the enzyme has been shown to accept the atternate substrate beta-3-methylaspartyl
phosphate (
Shames et al., J Biol. Chem. 259:15331-15339 (1984)). The
Haemophilus influenzae enzyme has been the subject of enzyme engineering studies to alter substrate binding
affinities at the active site (
Blanco et al., Acta Crystallogr.D.Biol.Crystallogr. 60:1388-1395 (2004)). Other ASD genes/enzymes are found in
Mycobacterium tuberculosis (
Shafiani et al., J Appl Microbiol 98:832-838 (2005)),
Methanococcus jannaschii (
Faehnle et al., J Mol.Biol. 353:1055-1068 (2005)), and the infectious microorganisms
Vibrio cholera and
Helicobacter pylori (
Moore et al., Protein Expr.Pirof. 25:189-194 (2002)). A related enzyme is acetylglutamylphosphate reductase (EC 1.2.1.38), an enzyme
that naturally reduces acetylglutamylphosphate to acetylglutamate-5-semialdehyde,
found in
S. cerevisiae (
Pauwels et al., Eur.J Biochem. 270:1014-1024 (2003)),
B. subtilis (
O'Reilly et al., Microbiology 140 (Pt 5):1023-1025 (1994)),
E. coli (
Parsot et al., Gene. 68:275-283 (1988)), and other organisms. Additional phosphate reductase enzymes of
E.
coli include glyceraldehyde 3-phosphate dehydrogenase encoded by
gapA (
Branlant et al., Eur.J.Biochem 150:61-66 (1985)) and glutamate-5-semialdehyde dehydrogenase encoded by
proA (
Smith, et al., J.Bacteriol, 157:545-551 (1984b)). Genes encoding glutamate-5-semialdehyde dehydrogenase enzymes from
Salmonella typhimurium (
Mahan et al., J Bacteriol. 156:1249-1262 (1983)) and
Campylobacter jejuni (
Louie et al., Mol. Gen. Genet. 240:29-35 (1993)) were cloned and expressed in
E. coli.
Protein |
GenBank ID |
GI Number |
Organism |
asd |
NP_417891.1 |
16131307 |
Escherichia coli |
asd |
YP_248335.1 |
68249223 |
Haemophilus influenzae |
asd |
AAB49996 |
1899206 |
Mycobacterium tuberculosis |
VC2036 |
NP_231670 |
15642038 |
Vibrio cholera |
asd |
YP_M2301787.1 |
210135348 |
Helicobacter pylori |
ARG5,6 |
NP_010992.1 |
6320913 |
Saccharomyces cerevisiae |
argC |
NP_389001.1 |
16078184 |
Bacillus subtilis |
argC |
NP_418393.1 |
16131796 |
Escherichia coli |
gapA |
P0A9B2.2 |
71159358 |
Escherichia coli |
proA |
NP_414778.1 |
16128229 |
Escherichia coli |
proA |
NP_459319.1 |
16763704 |
Salmonella typhimurium |
proA |
P53000.2 |
9087222 |
Campylobacter jejuni |
[0338] 1.2.1.e Oxidoreductase (acid to aldehyde): Direct conversion of benzoate to benzaldehyde
or
p-toluate to
p-methylbenzaldehyde (Path B of Figures 10 and 11) is catalyzed, by a carboxylic acid
reductase. Exemplary enzymes include carboxylic acid reductase, alpha-aminoadipate
reductase and retinoic acid reductase. Carboxylic acid reductase (CAR) catalyzes the
magnesium, ATP and NADPH-dependent reduction of carboxylic acids to their corresponding
aldehydes (
Venkitasubramanian et al., J Biol.Chem. 282:478-485 (2007)). The natural substrate of this enzyme is benzoate and the enzyme exhibits broad
acceptance of aromatic substrates including
p-toluate (
Venkitasubramanian et al., Biocatalysis in Pharmaceutical and Biotechnology Industries.
CRC press (2006)). The enzyme from
Nocardia iowensis, encoded by
car, was cloned and functionally expressed in
E. coli (
Venkitasubramanian et al., J Biol.Chem. 282:478-485 (2007)). CAR requires post-translational activation by a phosphopantetheine transferase
(PPTase) that converts the inactive apo-enzyme to the active holo-enzyme (
Hansen et al., Appl.Environ.Microbiol 75:2765-2774 (2009)). Expression of the
npt gene, encoding a specific PPTase, improved activity of the enzyme. A similar enzyme
found in
Streptomyces griseus is encoded by the
griC and
griD genes. This enzyme has been indicated to convert 3-amino-4-hydroxybenzoic acid to
3-amino-4-hydroxybenzaidehyde, as deletion of either
griC or
griD led to accumulation of extracellular 3-acetylamino-4-hydroxybenzoic acid, a shunt
product of 3-amino-4-hydroxybenzoic acid metabolism (
Suzuki, et al., J. Antibiot. 60(6):380-387 (2007)). The
S. griseus PPTase is likely encoded by
SGR_665, as predicted by sequence homology to the Nocardia iowensis npt gene.
Gene |
GenBank Accession No. |
GI No. |
Organism |
car |
AAR91681.1 |
40796035 |
Nocardia iowensis |
npt |
ABI83656.1 |
114848891 |
Nocaredia iowensis |
griC |
YP_001825755.1 |
182438036 |
Streptomyces griseus |
griD |
YP_001825756.1 |
182438037 |
Streptomyces griseus |
SGR_665 |
YP_001822177.1 |
182434458 |
Streptomyces griseus |
[0339] An enzyme with similar characteristics, alpha-aminoadipate reductase (AAR, EC 1.2.1.31),
participates in lysine biosynthesis pathways in some fungal species. This enzyme naturally
reduces alpha-aminoadipate to alpha-aminoadipate semialdehyde. The carboxyl group
is first activated through the AT
P-dependent formation of an adenylate that is then reduced by NAD(P)H to yield the
aldehyde and AMP. Like CAR, this enzyme utilizes magnesium and is activated by a PPTase.
Enzymes for AAR and its corresponding PPTase are found in
Saccharomyces cerevisiae (
Morris et al., Gene 98:141-145 (1991)),
Candida albicans (
Guo et al., Mol. Genet. Genomics 269:271-279 (2003)), and
Schizosaccharomyces pombe (
Ford et al., Curr. Genet. 28:131-137 (1995)). The AAR from
S. pombe exhibited significant activity when expressed in
E. coli (
Guo et al., Yeast 21:1279-1288 (2004)). The AAR from
Penicillium chrysogenum accepts S-carboxymethyt-L-cysteine as an alternate substrate, but did not react with
adipate, L-glutamate or diaminopimelate (
Hijarrubia et al., J Biol. Chem. 278:8250-8256 (2003)). The gene encoding the
P. chrysogenum PPTase has not been identified to date and no high-confidence hits were identified
by sequence comparison homology searching.
Gene |
GenBank Accession No. |
GI No. |
Organism |
LYS2 |
AAA34747.1 |
171867 |
Saccharomyces cerevisiae |
LYS5 |
P50113.1 |
1708896 |
Saccharomyces cerevisiae |
LYS2 |
AAC02241.1 |
2853226 |
Candida albicans |
LYS5 |
AAO26020.1 |
28136195 |
Candida albicans |
Lys1p |
P40976.3 |
13124791 |
Schizosaccharomyces pombe |
Lys7p |
Q10474.1 |
1723561 |
Schizosaccharomyces pombe |
Lys2 |
CAA74300.1 |
3282044 |
Penicillium chrysogenum |
[0340] 2.3.1.a Acyltransferase (phosphotransacylase): An enzyme with phosphotransbenzoylase
activity is used to interconvert benzoyl-CoA and (benzoyloxy)phosphonate (Path F of
Figure 10). A similar enzyme with phosphotrans-
p-methylbenzoylase activity interconverts
p-methylbenzoyl-CoA and (
p-methythenzoyloxy) phosphonate (Path F of Figure 11). Exemplary phosphate-transferring
acyltransferases include phosphotransacetylase (EC 2.3.1.8) and phosphotransbutyrylase
(EC 2.3.1.19). The
pta gene from
E. coli encodes a phosphotransacetylase that reversibly converts acetyl-CoA into acetyl-phosphate
(
Suzuki, Biochim. Biophys.Acta 191:559-569 (1969)). This enzyme can also convert propionyl-CoA propionylphosphate (
Hesslinger et al., Mol.Microbiol 27:477-492 (1998)). Other phosphate acetyltransferases that exhibit activity on propionyl-CoA are
found in
Bacillus subtilis (
Rado et al., Biochim.Biophys.Acta 321:114-125 (1973)),
Clostridium kluyveri (Stadtman, 1:596-599 (1955)), and
Thermotoga maritima (
Bock et al., J Bacteriol. 181:1861-1867 (1999)), Similarly, the
ptb gene from
C. acetobutylicum encodes phosphotransbutyrylase, an enzyme that reversibly converts butyryl-CoA into
butyryl-phosphate (
Wiesenborn et al., Appl Environ.Microbiol 55:317-322 (1989);
Walter et al., Gene 134:107-111 (1993)). Additional
ptb genes are found in butyrate-producing bacterium L2-50 (
Louis et al., J. Bacteriol. 186:2099-2106 (2004)) and
Bacillus megaterium (
Vazquez et al., Curr.Microbiol 42:345-349 (2001)).
Gene |
GenBank Accession No. |
GI No. |
Organism |
pta |
NP_416800.1 |
71152910 |
Escherichia coli |
pta |
P39646 |
730415 |
Bacillus subtilis |
pta |
A5N801 |
146346896 |
Clostridium kluyveri |
pta |
Q9X0L4 |
6685776 |
Thermotoga maritima |
ptb |
NP_349676 |
34540484 |
Clostridium acetobutylicum |
ptb |
AAR19757.1 |
38425288 |
butyrate-producing bacterium L2-50 |
ptb |
CAC07932.1 |
10046659 |
Bacillus megaterium |
[0341] 2.7.2.a Phosphotransferase, carboxyl group acceptor (kinase): Kinase or phosphotransterase
enzymes transform, carboxylic acids to phosphonic acids with concurrent hydrolysis
of one ATP. Such an enzyme is used to convert benzoate to (benzoyloxy)phosphonate
(Figure 10, Path H) and
p-toluate to (
p-methylbenzoyloxy)ptiosphonate (Figure 11, Path H). These exact transformations have
not been demonstrated to date. Exemplary enzymes include butyrate kinase (EC 2.7.2.7),
isobutyrate kinase (EC 2.7.2.14), aspartokinase (EC 2.7.2.4), acetate kinase (EC 2.7.2.1)
and gamma-glutamyl kinase (EC 2.7.2.11). Butyrate kinase catalyzes the reversible
conversion of butyryl-phosphate to butyrate during acidogenesis in
Clostridial species (
Cary et al., Appl. Environ. Microbiol 56:1576-1583 (1.990)). The
Clostridium acetobutylicum enzyme is encoded by either of the two
buk gene products (
Huang et al., J Mol. Microbiol Biotechnol 2:33-38 (2000)). Other butyrate kinase enzymes are found in
C. butyricum and
C. tetanomorphum (
TWAROG et al., J Bacteriol. 86:112-117 (1963)). A related enzyme, isobutyrate kinase from
Thermotoga maritima, was expressed in
E. coli and crystallized (
Diao et al., J Bacteriol. 191:252,1-2529 (2009);
Diao et al., Acta Crystallogr.D.Biol.Crystallogr. 59:1100-1102(2003)). Aspartokinase catalyzes the AT
P-dependent phosphorylation of aspartate and participates in the synthesis of several
amino acids. The aspartokinase III enzyme in
E. coli, encoded by
lysC, has a broad substrate range and the catalytic residues involved in substrate specificity
have been elucidated (
Keng et al., Arch.Biochem.Biophys. 335:73-81 (1996)). Two additional kinases in
E. coli are acetate kinase and gamma-glutamyl kinase. The
E. coli acetate kinase, encoded by
ackA (
Skarstedt et al., J.Biol.Chem. 251:6775-6783 (1976)), phosphorylates propionate in addition to acetate (
Hesslinger et al., Mol.Microbiol 27:477-492 (1998)). The
E. coli gamma-glutamyl kinase, encoded by
proB (
Smith et al., J Bacteriol. 157:545-551 (1984a)), phosphorylates the gamma carbonic acid group of glutamate.
Gene |
GenBank ID |
GI Number |
Organism |
buk1 |
NP_349675 |
15896326 |
Clostidium acetobutylicum |
buk2 |
Q97II1 |
20137415 |
Clostridium acetobutylicum |
buk2 |
Q9X278.1 |
6685256 |
Thermotoga maritima |
lysC |
NP_418448.1 |
16131850 |
Escherichia coli |
ackA |
NP_416799.1 |
16130231 |
Escherichia coli |
prokB |
NP_414777.1 |
16128228 |
Escherichia coli |
[0342] 2.8.3.a CoA transferase (10/11 A). CoA transferases catalyze the reversible transfer
of a CoA moiety from one molecule to another. Path A of Figure 10 is catalyzed by
an enzyme with benzoyt-CoA transferase activity. In this transformation, benzoyl-CoA
is formed from benzoate by the transfer of the CoA group from a CoA donor such as
acetyl-CoA, succinyl-CoA or others.
p-Methylbenzoyl-CoA transferase catalyzes a similar reaction from
p-toluate in Path A of Figure 11. Exemplary CoA transferase enzymes that react with
similar substrates include cinnamoyl-CoA transferase (EC 2.8.3.17) and benzylsuccinyl-CoA
transferase. Cinnamoyl-CoA transferase, encoded by
fldA in
Clostridium sporogenes, transfers a CoA moiety from cinnamoyl-CoA to a variety of aromatic acid substrates
including phenylacetate, 3-phetiylpropionate 4-phenylbutyrate (
Dickert et al., Eur.J Biochem. 267:3874-3884 (2000)). Benzylsuccinyl-CoA transferase utilizes succinyl-CoA or maleyl-CoA as the CoA
donor, forming benzylsuccinyl-CoA from benzylsuccinate. This enzyme was characterized
in the denitrifying bacteria
Thauera aromatica, where it is encoded by
bbsEF (
Leutwein et al., J Bacteriol. 183:4288-4295 (2001)).
Gene |
GenBank Accession No. |
GI No. |
Organism |
fldA |
AAL18808.1 |
16417587 |
Clostridium sporogenes |
bbsE |
AAF89840.1 |
9622535 |
Thauera aromatica |
bbsF |
AAF89841.1 |
9622536 |
Thauera aromatica |
[0343] Additional CoA transferase enzymes with diverse substrate ranges include succinyl-CoA
transferase, 4-hydroxybutyryl-CoA transferase, butyryl-CoA transferase, gtutaconyl-CoA
transferase and aectoacetyl-CoA transferase. The gene products of
cat1, cat2, and
cat3 of
Clostridium kluyveri have been shown to exhibit succinyl-CoA, 4-hydroxybutyryl-CoA, and butyryl-CoA transferase
activity, respectively (
Seedorf et al., Proc.Natl.Acad.Sci U.S.A 105-2128-2133 (2008);
Sohling et al., J Bacteriol. 178:871-880 (1996b)). Similar CoA transferase activities are also present in
Trichomonas vaginalis (
van Grinsven et al., J.Biol.Chem. 283:1411-1418 (2008)) and
Trypanosoma brucei (
Riviere et al., J.Biol.Chem. 279:45337-45346 (2004)). The glutaconyl-CoA-transferase (EC 2.8.3.12) enzyme from anaerobic bacterium
Acidaminococcus fermentans reacts with glutaconyl-CoA and 3-butenoyt-CoA (
Mack et al., Eur.J.Biochem. 226:41-51 (1994)). The genes encoding this enzyme are
gctA and
gctB. This enzyme has reduced but detectable activity with other CoA derivatives including
glutaryl-CoA, 2-hydroxyglutaryl-CoA, adipyl-CoA, crotonyl-CoA and acrylyl-CoA (
Buckel et al., Eur.J Biochem. 118:315-321 (1981)). The enzyme has been cloned and expressed in
E. coli (
Mack et al., Eur.J.Biochem. 226:41-51 (1994)). Glutaconate CoA-transferase activity has also been detected in
Clostridium sporophaeroides and
Clostridium symbiosum. Acetoacetyl-CoA transferase utilizes acetyl-CoA as the CoA donor. This enzyme is
encoded by the
E. coli atoA (alpha subunit) and
atoD (beta subunit) genes (
Korolev et al., Acta Crystallogr.D.Biol.Crystallogr. 58:2116-2121 (2002);
Vanderwinkel et al., Biochem.Biophys.Res.Commun. 33:902-908 (1968)). This enzyme has a broad substrate range (
Sramek et al., Arch.Biochem.Biophys. 171:14-26 (1975)) and has been shown to transfer the CoA moiety from acetyl-CoA to a variety of substrates,
including isobutyrate (
Matthies et al., Appl Environ.Microbiol 58:1435-1439 (1992)), valerate (
Vanderwinkel et al., Biochem.Biophys.Res.Commun. 33:902-908 (1968)) and butanoate (
Vanderwinkel et al., Biochem.Biophys.Res.Commun. 33:902-908 (1968)). Similar enzymes exist in
Corynebacterium glutamicum ATCC 13032 (
Duncan et al., Appl.Environ.Microbiol 68:5186-5190 (2002)),
Clostridium acetobutylicum (
Cary et al., Appl.Environ.Microbiol 56:1576-1583 (1990);
Wiesenborn et al., Appl.Environ.Microbiol 55:323-329 (1989)), and
Clostridium saccharoperbutylacetonicum (
Kosaka et al., Biosci.Biotechnol Biochem. 71:58-68 (2007)).
Gene |
GenBank Accession No. |
GI No. |
Organism |
cat1 |
P38946.1 |
729048 |
Clostridium kluyveri |
cat2 |
P38942.2 |
172046066 |
Clostridium kluyveri |
cat3 |
EDK35586.1 |
146349050 |
Clostridium kluyveri |
TVAG_395550 |
XP_001330176 |
123975034 |
Trichomonas vaginalis G3 |
Tb11.02.0290 |
XP_828352 |
71754875 |
Trypanosoma brucei |
gctA |
CAA57199.1 |
559392 |
Acidaminococcus fermentans |
gctB |
CAA57200.1 |
559393 |
Acidaminococcus fermentans |
gctA |
ACJ24333.1 |
212292816 |
Clostridium symbiosum |
getB |
ACJ24326.1 |
212292808 |
Clostridium symbiosum |
atoA |
P76459.1 |
2492994 |
Escherichia coli K12 |
atoD |
P76458.1 |
2492990 |
Escherichia coli K12 |
actA |
YP_226809.1 |
62391407 |
Corynebacterium glutamicum |
cg0592 |
YP_224.801.1 |
62389399 |
Corynebacterium glutamicum |
tfA |
NP_149326.1 |
15004866 |
Clostridium acetobutylicum |
ctfB |
NP_149327.1 |
15004867 |
Clostridium acetobutylicum |
ctfA |
AAP42564.1 |
31075384 |
Clostridium saccharoperbutylacetonicum |
ctfB |
AAP42565.1 |
31075385 |
Clostridium saccharoperbutylacetonicum |
[0344] 3.1.2.a CoA hydrolase (10/11 A): Benzoyl-CoA and
p-methylbenzoyl-CoA can be hydrolyzed to their corresponding acids by CoA hydrolases
or thioesterases in the EC class, 3.1.2 (Path A of Figures 10 and 11). Exemplary CoA
thioesters that hydrolyze benzoyl-CoA and/or similar substrates include 4-hydroxybenzoyl-CoA
hydrolase (EC 3.1.2.23) and phenylglyoxal-CoA hydrolase (EC 3.1.2.25). The
Azoarcus evansii gene
orf1 encodes an enzyme with benzoyl-CoA hydrolase activity that participates in benzoate
metabolism (
Ismail, Arch.Microbiol 190:451-460 (2008)). This enzyme, when heterologously expressed in
E. coli, demonstrated activity on a number of alternate substrates. Additional benzoyl-CoA
hydrolase enzymes were identified in benzonate degradation gene clusters of
Magnetospirillum magnetotsacticum, Jannaschia sp. CCS1 and
Sagittula stellata E-37 by sequence similarity (
Ismail, Arch.Microbiol 190:451-460 (2008)). The 4-hydroxybenzoyl-CoA hydrolase of
Pseudomonas sp. CBS3 accepts benzoyl-CoA and
p-methylbenzoyl-CoA as substrates and has been heterologously expressed and characterized
in
E. coli (
Song et al., Bioorg.Chem. 35:1-10 (2007)). Additional enzymes with demonstrated benzoyl-CoA hydrolase activity include the
palmitoyl-CoA hydrolase of
Mycobacterium tuberculosis (
Wang et al., Chem.Biol. 14:543-551 (2007)) and the acyl-CoA hydrolase of
E. coli encoded by
entH (
Guo et al., Biochemistry 48:172-1722 (2009)).
Gene |
GenBank Accession No. |
GI No. |
Organism |
orf1 |
AAN39365.1 |
23664428 |
Azoarcus evansii |
Magn03011230 |
ZP_00207794 |
46200680 |
Magnetospirillum magnetotacticum |
Jann_0674 |
YP_508616 |
89053165 |
Jannaschia sp. CCS1 |
SSE37_24444 |
ZP_01745221 |
126729407 |
Sagittula stellata |
EF569604.1:4745..5170 |
ABQ44580.1 |
146761194 |
Pseudomonas sp.CBS3 |
Rv0098 |
NP_214612.1 |
15607240 |
Mycobacterium tuberculosis |
entH |
AAC73698.1 |
1786813 |
Escherichia, coli |
[0345] Several CoA hydrolases with broad substrate ranges are suitable enzymes for hydrolyzing
benzoyl-CoA and/or
p-methylbenzoyl-CoA. For example, the enzyme encoded by
acot12 from
Rattus norvegicus brain (
Robinson et al., Biochem.Biolphys.Res.Commun. 71:959-965 (1976)) can react with butyryl-CoA, hexanoyl-CoA and malonyl-CoA. The human dicarboxylic
acid thioesterase, encoded by
acot8, exhibits activity on glutaryl-CoA, adipyl-CoA, suberyl-CoA, sebacyl-CoA, and dodecanedioyl-CoA
(
Westin et al., J.Biol.Chem. 280:38125-38132 (2005)). The closest
E. coli homolog to this enzyme,
tesB, can also hydrolyze a range of CoA thiolesters (
Naggert et al., J Biol Chem 266:11044-11050 (1991)). A similar enzyme has also been characterized in the rat liver (
Deana R., Biochem Int 26:767-773 (1992)).
Gene name |
GI# |
GenBank Accession # |
Organism |
acot12 |
18543355 |
NP_570103.1 |
Rattus norvegicus |
tesB |
16128437 |
NP_414986 |
Escherichia coli |
acot8 |
3191970 |
CAA15502 |
Homo sapiens |
acot8 |
51036669 |
NP_570112 |
Rattus norvegicus |
tesA |
16128478 |
NP_415027 |
Esceherichia coli |
ybgC |
16128711 |
NP_415264 |
Escherichia coli |
paaI |
16129357 |
NP_415914 |
Escherichia coli |
ybdB |
16128580 |
NP_415129 |
Escherichia coli |
[0346] 4.1.1a. Carboxy-lyase (6/7 E): Decarboxylase enzymes in the EC class 4.1.1 are used
to convert benzoate to benzene (Path E of Figure 10) and
p-toluate to toluene (Path E of Figure 11). Exemplary enzymes that react with these
or similar substrates include benzene carboxylase, vanillate decarboxylase, cinnamate
decarboxylase, aminobenzoate decarboxylase and a variety of hydroxybenzoate decarboxylases.
Decarboxylase enzymes can be oxi dative or nonoxidative, depending on the cofactors
utilized (
Lupa et al., Genomics 86:342-351 (2005a)). An enzyme predicted to have benzoate carboxylase activity was identified in a
Clostridia bacterium enrichment culture clone BF (
Abu et al., Environ. Miicrobiol (2010)).
Gene |
GenBank Accession No. |
Gl No. |
Organism |
abcA |
ADJ94002.1 |
300245889 |
Clostridia bacterium enrichment culture clone BF |
abcD |
ADJ94001.1 |
300245887 |
Clostridia bacterium enrichment culture clone BF |
[0347] A number of characterized decarboxylases with demonstrated activity on hydroxylated
aromatics such as 4-hydroxybenzoate, 2,3-dihydroxybelizoa.te, 3,4-dihydroxybenzoate,
2,6-dihydroxybenzoate and 4,5-dihydroxyphthalate can also exhibit activity on alternate
substrates such as
p-toluate or benzoate. Exemplary hydroxybeazoate decarboxylase enzymes include the
4,5-dihydroxyphthalate decarboxylase of
Comamonas testosteroni (
Nakazawa et al., Appl.Environ.Microbiol 36:264-269 (1978)), the 2,3-dihydroxybenzoate decarboxylase of
Aspergillus niger (
Kamath et al., Biochem. Biophys. Res. Commun. 145:586-595 (1987)) and the 3-octaprenyl-4-hydroxybenzoate decarboxylase of
E. coli (
Zhang et al., J Bacteriol. 182:6243-6246 (2000)). Exemplary 4-hydroxybenzoate decarboxylases are encoded by
shdBD and
ubiD of
Sedimentibacter hydroxybenzoicus (formerly
Clostridium hydroxybenzoicum) and
ubiD of
Enterobacter cloacae P240 (
Matsui et al., Arch.Microbiol 186:21-29 (2006a);
He et al., Eur. J Biochem. 229:77-82 (1995)). The 4-hydroxybenzoate decarboxylase from the facultative anaerobe,
Enterobacter cloacae,
encoded by ubiD, has been tested for activity on multipte substrates and was shown to be induced by
both 4-hydroxybenzoic acid and 4-aminobenzoic acid (
Matsui et al., Arch.Microbiol 186:21-29 (2006b)). The
bsdBCD genes of
Bacillus siibtilis encode a reversible non-oxidative 4-hydroxybenzoate/vanillate decarboxylase (
Lupa et al., Can.J Microbiol 54:75-81 (2008)). This enzyme was heterologousty expressed in
E.
coli. Similar decarboxylases have been indicated in several other organisms (
Lupa et al., Genomics 86:342-351 (2005b)) and genes for some of these are listed below.
Gene |
GenBank Accession No. |
GI No. |
Organism |
phtD |
Q59727.1 |
3914354 |
Comamonas testosteroni |
dhbD |
CAK48106.1 |
134075758 |
Aspergillus niger |
ubiD |
NP_418285.1 |
16131689 |
Escherichia coli |
shcD |
AAY67851.1 |
67462198 |
Sedimentbacter hydroxybenzoicus |
shdB |
AAY67850.1 |
67462197 |
Sedimentibacter hydroxybenzoicus |
ubiD |
AAD50377.1 |
5739200 |
Sedimentibacter hydroxybenzoicus |
ubiD |
BAE97712.1 |
110331749 |
Enterobacter cloacae P240 |
bsdB |
CAB12157.1 |
2632649 |
Bacillus subtilis |
bsdC |
CAB12158.1 |
2632650 |
Bacillus subtilis |
bsdD |
CAB12159.1 |
2632651 |
Bacillus subtilis |
STM292 |
NP_461842.1 |
16766227 |
Salmonella typhimurium LT2 |
STM2922 |
NP_461843.1 |
16766228 |
Salmonella typhimurium LT2 |
STM2923 |
NP_461844.1 |
16766229 |
Salmonella typhimurium LT2 |
kpdB |
YP_002236894.1 |
206580833 |
Klesbiella pneumoniae 342 |
kpdC |
YP_002236895.1 |
206576360 |
Klesbiella pneumoniae 342 |
kpdD |
YP_002236896.1 |
206579343 |
Klesbiella pneumoniae 342 |
pad1 |
NP_311620.1 |
15832847 |
Escherichia coli O157 |
yclC |
NP_311619.1 |
15832846 |
Escherichia coli O157 |
yclD |
NP_311618.1 |
15832845 |
Escherichia coli O157 |
[0348] An additional class of decarboxylases has been characterized that the decarboxylation
of cinnamate (phenylacrylate) and substituted cinnamate derivatives. These enzymes
are common in a variety of organisms and specific genes encoding these enzymes that
have been cloned and expressed in
E. coli include
pad 1 from
Saccharomyces cerevisae (
Clausen. et al., Gene 142:107-112 (1994)),
pdc from
Lactobacillus plantarum (Barthelmebs et al., 67:1063-1069 (2001);
Qi et al., Eng 9:268-276 (2007); et al.,
J. Agric. Food Chem. 56:3068-3072 (2008)),
pofk (
pad) from
Klebsiella oxytoca(
Uchiyama et al., Biosci.Biotecnol.Biochem. 72:116-123 (2008);
Hasbidoko et al., Biosci.Biotech.Biochem. 58:217-218 (1994)),
Pedicoccus pentosaceus (Barthelmebs et al., 67:1063-1069 (2001)), and
padC from
Bacillus subtilis and
Bacillus pumilus (Shingler et al., 174:711-724 (1992)). A ferulic acid decarboxylase from
Pseudomonas fluorescens also has been purified and characterized (
Huang et al., J.Bacteriol. 176:5912-5918 (1994)). Enzymes in this class have been shown to be stable and do not require either exogenous
or internally bound co-factors, thus making these enzymes suitable for biotransformations
(
Sariaslani; Annu. Rev. Microbiol. 61:5-1-69 (2007)).
Protein |
GenBank ID |
GI Number |
Organism |
pad1 |
AAB64980.1 |
1165293 |
Saccharomyces cerevisae |
pdc |
AAC45282.1 |
1762616 |
Lactobacillus plantarum |
pad |
BAF65031.1 |
149941608 |
Klebsiella oxytoca |
padC |
NP_391320.1 |
16080493 |
Bacillus subtilis |
pad |
YP_804027.1 |
116492292 |
Peclicoccus pentosaceus |
pad |
CAC18719.1 |
11691810 |
Bacillus pumilus |
[0349] 4.1.99.a Decarbonylase: A decarbonylase enzyme is to convert benzaldehyde to benzene
(Path C of Figure 10) and
p-methylbenzaldehyde to toluene (Path C of Figure 11). Decarbonylase enzymes catalyze
the final step of alkane biosynthesis in plants, mammals, insects and bacteria (
Dennis et al., Arch.Biochem.Biophys.287:268-275 (1991)). Non-oxidative decarbonylases transform aldehydes into alkanes with the concurrent
release of CO. Exemplary decarbonylase enzymes include octadecanal decarbonylase (EC
4.1.99.5), sterol desaturase and fatty aldehyde decarbonylase. The CER1 gene of Arabidopsis
thaliana encodes a fatty acid decarbonylase involved in epicuticular wax formation
(
US 6,437,218). Additional fatty acid decarbonylases are found in
Medicago truncatula,
Vitis vinifera and
Oryza sativa (
US Patent Application 2009/0061493). A cobalt-porphyrin containing decarbonylase was purified and characterized in the
algae
Botryococcus braunii; however, no gene has been associated with this activity to date (
Dennis et al., Proc.Natl.Acad. Sci. U.S.A 89.-5306-5310 (1992)). A copper-containing decarbonylase from
Pisum sativum was also purified and characterized, but is not yet associated with a gene (
Schneider-Belhaddad et al., Arch. Biochem.Biophys. 377:341-349 (2000)).
Gene |
GenBank Accession No. |
GI No. |
Organism |
CER1 |
NP_850932 |
145361948 |
Arabidopsis thaliana |
MtrDRAFT_AC153128g2v2 |
ABN07985 |
124359969 |
Medicago truncatula |
VITISV_029045 |
CAN60676 |
147781102 |
Vitis vinifera |
OSJNBa0004N05.14 |
CAE03390.2 |
38345317 |
Oryza sativa |
[0350] Alternately, an oxidative decarbonylase can convert an aldehyde into an alkane. Oxidative
decarbonylases are cytochrome P450 enzymes that utilize NADPH and O
2 as cofactors and release CO
2, water and NADP
+. This activity was demonstrated in the
CYP4G2vl and
CYP4G1gene products
of Musca domestica and
Drosophila, melanogaster (
US Patent Application 2010/0136595). Additional enzymes with oxidative decarbonylase activity can be identified by sequence
homology in other organisms such as
Mamestra brassicae, Helicoverpa zea and
Acyrthosiphon pisum.
Protein |
GenBank ID |
GI Number |
Organism |
CYP4G2v1 |
ABV48808.1 |
157382740 |
Musca domestica |
CYP4G1 |
NP_525031.1 |
17933498 |
Drosophila melanogaster |
CYP4G25 |
BAD81026.1 |
56710314 |
Antheraea yamamai |
CYP4M6 |
AAM54722.1 |
21552585 |
Helicoverpa zea |
LOC100164072 |
XP_001944205.1 |
193650239 |
Acyrthosiphon pisum |
[0351] 6.2.1.a.Acid-thiol ligase (8A, 11B): The AT
P-dependent activation of benzoate to benzoyl-CoA or
p-toluate to
p-methylbenzoyl-CoA (Path A of Figures 10 and 11) is catalyzed by a CoA synthetase
or acid-thiot ligase. AM
P-forming CoA ligases activate the aromatic acids to their corresponding CoA derivatives,
whereas AD
P-forming CoA ligases are generally reversible. Exemplary AM
P-forming benzoyl-CoA ligases from
Thauera aromatica and
Azoarcus sp. strain CIB have been characterized (
Lopez Barragan et al., J Bacteriol. 186:5762-5774(2004);
Schuhle et al., J.Bacteriol. 185:4920-4929 (2003)). Alternately, AM
P-forming CoA ligases that react with structurally similar substrates can have activity
on benzoate or
p-toluate, The AM
P-forming cyclohexanecarboxylate CoA-ligase from
Rhodopseudomonas palustris, encoded by
aliA, is well-characterized, and alteration of the active site has been shown to impact
the substrate specificity of the enzyme (
Samanta et al., Mol.Microbiol 55:1151-1159 (2005)). This enzyme also functions as a cyclohex-1-ene-1-carboxylate CoA-ligase during
anaerobic benzene ring degradation (
Egland et al., Proc.Natl.Acad.Sci U.S.A 94:6484-6489 (1997)). Additional exemplary CoA ligases include two characterized phenylacetate-CoA ligases
from
P.
chrysogenum (
Lamas-Maceiras et al., Biochem.J 395. 147-155 (2006);
Wang et al., Biochem.Biophys.Res.Commun. 360:453-458 (2007));
Wang et al., Biochem. Biophys. Res. Commun. 360:453-458 (2007)), the phenylacetate-CoA ligase from
Pseudomonas putida(
Martinez-Blanco et al.,J Biol.Chem. 265,7084-7090 (1990)), and the 6-carboxyhexanoate-CoA ligase from
Bacillus subtilis (
Bower et al., J Bacteriol. 178:4122-4130 (1996)).
Gene |
GenBank Accession No. |
GI No. |
Organism |
bclA |
Q8GQN9.1 |
75526585 |
Thauera aromatica |
bzdA |
AAQ08820.1 |
45649073 |
Azoarcus sp. strain CIB |
ali A |
AAC23919 |
2190573 |
Rhodopseudomonas palustris |
phl |
CAJ15517.1 |
77019264 |
Penicillium chrysogenum |
phlB |
ABS19624.1 |
152002983 |
Penicillium chrysogenum |
paaF |
AAC24333.2 |
22711873 |
Pseudomonas putida |
bioW |
NP_390902.2 |
50812281 |
Bacillis subtilis |
[0352] ADP-forming CoA ligases catalyzing these exact transformations have not been characterized
to date; however, several enzymes with broad substrate specificities have been described
in the literature. The ADP-forming acetyl-CoA synthetase (ACD, EC 6.2.1.13) from
Archaeoglobus fulgidus, encoded by AF1211, was shown to operate on a variety of linear and branched-chain
substrates including isobutyrate, isopentanoate, and fumarate (
Musfeldt et al., J Bacteriol. 184:636-644 (2002)). A second reversible ACD in
Archaeoglobus fulgidus,encoded by
AF1983, was also indicated to have a broad substrate range with high activity on aromatic
compounds phenylacetate and indoleacetate (Musfeldt et al.,
supra).The enzyme from
Haloarcula marismortui, annotated as a succinyl-CoA synthetase, accepts propionate, butyrate, and branched-chain
acids (isovalerate and isobutyrate) as substrates, and was shown to operate in the
forward and reverse, directions (
Brasen et al., Arch. Microbiol 182:277-287 (2004)). The ACD encoded by
P4E3250 from hyperthermophilic crenarchaeon
Pyrobaculum aerophilum showed the broadest substrate range of all characterized ACDS, reacting with acetyl-CoA,
isobutyryl-CoA (preferred substrate) and phenyhcetyl-CoA and
Schonheit, Arch. Microbiol 182:277-287 (2004)). Directed evolution or engineering can be used to modify this enzyme to operate
at the physiological temperature of the host organism. The enzymes from
A. fulgidus, H. marismortui and
P. aerophilum have all been cloned, functionally expressed, and characterized in
E. coli (
Brasen and Schonheit, Arch. Microbiol 182:277-287 (2004);
Musfetdt and Schonheit, J Bacteriol. 184:636-644 (2002)). An additional enzyme is encoded. by
sucCD in
E. coli, which naturally catalyzes the formation of succinyl-CoA from succinate with the concomitant
consumption of one ATP, a reaction which is reversible
in vivo (
Buck et al., Biochemistry 24:6245-6252 (1985)). The acyl CoA ligase from
Pseudomonas putida has been indicated to work on several aliphatic substrates including acetic, propionic,
butyric, valeric, hexanoic, heptanoic, and octanoic acids and on aromatic compounds
such as phenylacetic and phenoxyncetic (
Fernandez-Valverde et al., Appl. Environ. Microbiol. 59-1149-1154 (1993)). A related enzyme, malonyl CoA synthetase (63.4.9) from
Rhizobium leguminosarum could convert, several diacids, namely, ethyl-, propyl-, allyl-, isopropyl-, dimethyl-,
cyclopropyl-, cyclopropylmethylene-, cyclobutyl-, and benzyl-malonate into their corresponding
monothioesters (
Pohl et al., J.Am.Chem.Soc. 123-5822-5823 (2001)).
Gene |
GenBank Accession No. |
GI No. |
Organism |
AF1211 |
NP_070039.1 |
11498810 |
Archaeoglobus fulgidus |
AF1983 |
NP_070807.1 |
11499565 |
Archaeoglobus fulgidus |
SCS |
YP_135572.1 |
55377722 |
Haloarcula marismortui |
PAE3250 |
NP_560604.1 |
18313937 |
Pyrobaculum aerophilum str. |
|
|
|
IM2 |
sucC |
NP_415256.1 |
16128703 |
Escherichia coli |
sucD |
AAC73823.1 |
1786949 |
Escherichia coli |
paaF |
AAC24333.2 |
22711873 |
Pseudomonas putida |
matB |
AAC83455.1 |
3982573 |
Rhizobium leguminosarum |
EXAMPLE VIII
Pathways to 2,4-pentadienoate from pyruvate, ornithine and alanine
[0353] This example shows pathways from pyruvate, ornithine and alanine to 2,4-pentadienoate.
[0354] Figure 12 shows the conversion of pyruvate to 2,4-pentadienoate. This conversion
can be accomplished in four enzymatic steps. Pyruvate and acetaldehyde are first condensed
to 4-hydroxy-2-oxovaterate by 4-hydroxy-2-ketovalerate aldolase (Step A of Figure
12). The 4-hydroxy-2-oxovalerate product is next dehydrated to 2-oxopentenoate (Step
B of Figure 12). Subsequent reduction and dehydration of 2-oxopentenoate yields 2,4-pentadienoate
(Steps C/D of Figure 12).
[0355] Figure 13 shows pathways from alanine or ornithine to 2,4-pentadienoate. In Step
A of Figure 13, alanine and acetyl-CoA are joined by AKP thiolase to form AKP. In
one pathway, AKP is deaminated to acetylacrtate (Step B). The 4-oxo group of acetylacrylate
is then reduced and dehydrated to 2,4-pentadienoate (Steps C/D). In all alternative
pathway, AKP is converted to 2,4-dioxopentanoate by an aminotransferase or dehydrogenase
(Step E). Reduction of the 2- or 4-oxo group of 2,4-dioxopentanoate 2-hydroxy-4-oxopentanoate
(Step H) or 4-hydroxy-2-oxovaterate (Step K), respectively. 4-Hydroxy-2-oxovaterate
can alternately be formed by the reduction of AKP to 2-amino-4-hydroxypentanoate (Step
J) followed by transamination or oxidative deamination (Step L). Once formed, 4-hydroxy-2-oxovalerate
can be converted to 2,4-pentadienoate in three enzymatic steps as shown in Figure
12 (Steps B/C/D of Figure 12). The 2-hydroxy4-oxopentanoate intermediate can undergo
dehydration to acetylacrylate (Step F) followed by reduction and dehydration (Steps
C/D).
[0356] An alternate entry point into the pathways from AKP shown in Figure 13 is ornithine.
An ornithine aminomutase is first required to convert ornithine to 2,4-diaminopentanoate
(Step M). The 2,4-diaminopentanoate intermediate is then converted to AKP by transamination
or oxidative deamination (Step N).
[0357] It is understood, that either the D- or L- stereoisomer of alanine or ornithine can
serve serve as the precursor or intermediate to a 2,4-pentadienoate pathway shown
in Figure 13. The D- and L- stereoisomers of alanine or ornithine are readily interconverted
by alanine racemase or ornithine racemase enzymes.
[0358] Enzymes for catalyzing the transformations shown in Figures 5 and 6 are categorized
by EC number (Table 2) and described further below.
Label |
Function |
Step |
1.1.1.a |
Oxidoreductase (oxo to alcohol) |
1C, 2C/H/K/J |
1.4.1.a |
Oxidoreductase (aminating/deaminating) |
2E/L/N |
2.6.1.a |
Aminotransferase |
2E/L/N |
4.1.3.a |
Lyase |
1A |
4.2.1.a |
Hydro-lyase |
1B/D, 2D/F |
4.3.1a |
Ammonia-lyase |
2B |
5.1.1.a |
D/L Racemase |
2A/M |
5.4.3.a |
Aminomutase |
2M |
Other |
AKP thioloase |
2A |
[0359] 1.1.1.a Oxidoreductase (oxo to alcohol): A number of transformations in Figures 5
and 6 involve the reduction of a to an alcohol. In Step C of Figure 12, 2-oxopentenoate
is reduced to 2-hyroxypentenoate. A similar transformation is the reduction, of the
keto-acid 2,4-dioxopentanoate to its corresponding hydroxy-acid, 2-hydroxy-4-oxopentanoate
(Step H of Figure 13). Steps C, J and K of Figure 13 entail reduction of the 4-oxo
groups of AKP, 2,4-dioxopentanoate and acetylacrylate to their corresponding alcohols.
These transformations are catalyzed by oxidoreductase enzymes in the EC class 1.1.1.
[0360] Several exemplary alcohol dehydrogenases convert a ketone to an alcohol functional
group. Two such enzymes from
E. coli are encoded by malate dehydrogenase (
mdh) and lactate dehydrogenase (
ldhA). In addition, lactate dehydrogenase from
Ralstonia eutropha has been shown to demonstrate high activities on 2-ketoacids of various chain lengths
includings lactate, 2-oxobutyrate, 2-oxopentanoate and 2-oxoglutarate (
Steinbuchel et al., Eur.J.Biochem. 130:329-334 (1983)). Conversion of alpha-ketoadipate into alpha-hydroxyadipate is catalyzed by 2-ketoadipate
reductase, an enzyme found in rat and in human placenta (
Suda et al., Arch.Biochem.Biophys. 176:610-620 (1976);
Suda et al., Biochem.Biophys.Res.Commun. 77:586-591 (1977)). An additional candidate oxidoreductase is the mitochondrial 3-hydroxybutyrate
dehydrogenase (
bdh) from the human heart which has been cloned and characterized (
Marks et al.,J.Biol.Chem. 267:15459-15463 (1992)). Alcohol dehydrogenase enzymes of
C. beijerinckii (
Ismaiel et al., J.Bacteriol. 175:5097-5105 (1993)) and
T. brockii (
Lamed et al., Biochem.J. 195:183-190 (1981);
Peretz et al., Biochemistry. 28:6549-6555 (1989)) convert acetone to isopropanol. Methyl ethyl ketone reductase catalyzes the reduction
of MEK to 2-butanol. Exemplary MEK reductase enzymes can be found in
Rhodococctis ruber (
Kosjek et al., Biotechnol Boieng. 86:55-62 (2004)) and
Pyrococcus furiosus (
van der et al., Eur.J.Biochem. 268:3062-3068 (2001)),
Gene |
GenBank ID |
GI Number |
Organism |
mdh |
AAC76268.1 |
1789632 |
Escherichia coli |
IdhA |
NP_415898.1 |
16129341 |
Escherichia coli |
Idh |
YP_725182.1 |
113866693 |
Ralstonia eutropha |
bdh |
AAA58352.1 |
177198 |
Homo sapiens |
adh |
AAA23199.2 |
60592974 |
Clostridium, beijerinckii NRRL B593 |
adh |
P14941.1 |
113443 |
Thermoanaerobacter brockii HTD4 |
sadh |
CAD36475 |
21615553 |
Rhodococcus ruber |
adhA |
AAC25556 |
3288810 |
Pyrococcus furiosus |
[0361] 1.4.1.a Oxidoreductase (deaminating): Enzymes in the EC class 1.4.1 catalyze the
oxidative deamination of amino groups with NAD+, NADP+ or FAD as acceptor. Such all
enzyme is required to catalyze the oxidative deamination of AKP to 2,4-dioxopentanoate
(Figure 13, Step E), 2-amino-4-hydroxypentanoate to 4-hydroxy-2-oxovalerate (Figure
13, Step L) and 2,4-diaminopentanoate to AKP (Figure 13, Step N). The conversion of
2,4-diaminopentanoate to AKP (Step N of Figure 13) is catalyzed by 2,4-diaminopentanoate
dehydrogenase (EC 1.4.1.12). 2,4-Diaminopentanoate dehydrogenase enzymes have been
characterized in organisms that undergo anaerobic fermentation of ornithine, such
as the
ord gene product of
Clostridium sticklandii (
Fonknechten, J.Bacteriol. In Press: (2009)). Additional 2,4-diaminopentanoate dehydrogenase gene candidates can be inferred
in other organisms by sequence similarity to the
ord gene product. A related enzyme, 3,5-diaminohexanoate dehydrogenase (EC 1.4.1.11),
catalyzes the oxidative deamination of 3,5-diaminohexanoate to 5-amino-3-oxohexanoate.
The gene encoding this enzyme,
kdd, was recently identified in
Fusobacterium nucleatum (
Kreimeyer et al., J Biol.Chem. 282:7191-7197 (2007)). The enzyme has been purified characterized in other organisms that ferment lysine
but the genes associated with these enzymes have not been identified to date (
Baker et al., J Biol.Chem. 247:7724-7734 (1972);
Baker et al., Biochemistry 13:292-299 (1974)). Candidate in
Myxococcus xanthus, Porphyromonas gingivalis W83 and other sequenced organisms can be inferred by sequence homology.
Gene |
GenBank ID |
GI Number |
Organism |
ord |
CAQ42978.1 |
226885213 |
Clostridium sticklatidii |
Hore_21120 |
YP_002509852.1 |
220932944 |
Halothermothrix orenii |
CD0442 |
YP_001086913.1 |
126698016 |
Clostridium difficile |
kdd |
AAL93966.1 |
19713113 |
Fusobactenum nucleatum |
mxan_4391 |
ABF87267.1 |
108462082 |
Myxococcus xanthus |
pg_1069 |
AAQ66183.1 |
34397119 |
Pyrphyromonas gingivalis |
[0362] The substrates AKP and 2-amino-4-hydroxypentanoate (Steps E and L of Figure 13),
are similar to alpha-amino acids and may serve as alternate substrates for amino acid
dehydrogenase enzymes such as glutamate dehydrogenase (EC 1.4.1.2), leucine dehydrogenase
(EC 1.4.1.9), and aspartate dehydrogenase (EC 1.4.1.21). Glutamate dehydrogenase catalyzes
the reversible NAD(P)+ dependent conversion, of glutamate to 2-oxoglutarate. Exemplary
enzymes are encoded by
gdhA in
Escherichia coli (
McPherson et al., Nucleic.Acid Res. 11:5257-5266 (1983);
Korber et al., J.Mol.Biol. 234:1270-1273 (1993)),
gdh in
Thermotoga maritima (
Kort et al., Extremophiles 1:52-60 (1997);
Lebbink et al., J.Mol.Biol. 280:287-296 (1998);
Lebbink et al., J.Mol.Biol. 289:357-369 (1999)), and
gdhA1 in
Halobacterium salinarum, (
Ingoldsby et al., Gene. 349:237-244 (2005)). Additional glutamate dehydrogenase enzymes have been characterized in
Bacillus subtilis (
Khan et al., Biosci.Biotechnol Biochem. 69:186.1-1870 (2005)),
Nicotiana tabacum (
Purnell et al., Planta 222:167-180 (2005)),
Oryza sativa (
Abiko et al., Plant Cell Physiol 46:1724-1734 (2005)),
Haloferax mediterranei (
Diaz et al., Extremophiles. 10:105-115 (2006)) and
Halobactreium salinarum (
Hayden et al., FEMS Microbiol Lett. 211:37-41 (2002)). The
Nicotiana tabacum enzyme is composed of alpha and beta subunits encoded by
gdh1 and
gdh2 (
Purnell et al., Planta 222:167-180 (2005)). An exemplary leucine dehydrogenase is encoded by
ldh, of
Bacillus cereus. This enzyme reacts with a range of substrates including leucine, isoleucine, valine,
and 2-aminobutanoate (
Stoyan et al., J.Biotechnol 54:77-80 (1997);
Ansorge et al., Biotechnol Bioeng. 68:557-562 (2000)). The aspartate dehydrogenase from
Thermotoga maritime, encoded by
nadX, is involved in the biosynthesis of NAD (
Yang et al., J.Biol.Chem. 278-8804-8808 (2003)).
GGene |
GenBank ID |
GI Number |
Organism |
gdha |
P00370 |
118547 |
Escherichia coli |
gdh |
P96110.4 |
6226595 |
Thermotoga maritima |
gdhA1 |
NP_279651.1 |
15789827 |
Halobacterium salinarum |
rocG |
NP_391659.1 |
16080831 |
Bacillus subtilis |
gdh1 |
AAR11534.1 |
38146335 |
Nicotiana tabacum |
gdh2 |
AAR11535.1 |
38146337 |
Nicotiana tabacum |
GDH |
Q852M0 |
75243660 |
Oryza sativa |
GDH |
Q977U6 |
74499858 |
Haloferax mediterranei |
GDH |
P29051 |
118549 |
Halobactreium salinarum |
GDH2 |
NP_010066.1 |
6319986 |
Saccharomyces cerevisiae |
ldh |
P0A393 |
61222614 |
Bacillus cereus |
nadX |
NP_229443.1 |
15644391 |
Thermotoga maritima |
[0363] 2.6.1.a Aminotransferase: Several transformations in Figure 13 are catalyzed by aminotransferase
or transaminase enzymes, including the conversion of AKP to 2,4-dioxopentanoate (Step
E), 2-amino-4-hydroxypentanoate to 4-hydroxy-2-oxovalerate (Step L) and 2,4-diaminopentanoate
to AKP (Step N). Several aminotransferases convert amino acids and derivatives to
their corresponding 2-oxoacids. Such enzymes are particularly well-suited to catalyze
the transformations depicted in Steps E and L of Figure 13 (i.e. AKP aminotransferase
and 2-amino-4-hydroxypentanoate aminotransferase). Selection of an appropriate amino
acid aminotransferase for these transformations may depend on the stereochemistry
of the substrate. When the substrate is in the D-configuration, a D-amino acid aminotransferase
(EC 2.6.1.21) can be utilized, whereas the L-stereoisomer is the preferred substrate
of an L-amino acid aminotransferase such as aspartate aminotransferase (EC 2.6.1.1).
Aspartate aminotransferase naturally transfers an oxo group from oxaloacetate to glutamate,
forming alpha-ketoglutarate and aspartate. Aspartate aminotransferase activity is
catalyzed by, for example, the gene products of
aspC from
Escherichia coli (Yagi et al., 100-81-84(1979); Yagi et al., 113:83-89 (1985)),
AAT2 from
Saccharomyces cerevisiae (Yagi et al., 92:35-43 (1982)) and
ASP5 from
Arabidopsis thaliana (Kwok et al., 55:595-604 (2004); de la et al., 46:414-425 (2006);
Wilkie et al., Protein Expr.Purif. 12:381-389 (1998)). The enzyme from
Rattus norvegicus has been shown to transaminate alternate substrates such as 2-aminohexanedioic acid
and 2,4-diaminobutyric acid (
Recasens et al., Biochemistry 19:4583-4589 (1980)). Aminotransferases that work on other L-amino-acid substrates may also be able
to catalyze these transformation. Valine aminotransferase catalyzes the conversion,
of valine and pyruvate to 2-ketoisovalerate and alanine. The
E. coli gene,
avtA, encodes a similar enzyme (
Whalen et al., J.Bacteriol. 150:739-746 (1982)), which also catalyzes the transamination of α-ketobutyrate to generate α-aminobutyrate,
although the amino donor in this reaction has not been identified (
Whalen et al., J.Bacteriol. 158:571-574 (1984)). Another enzyme candidate is alpha-aminoadipate aminotransferase (EC 2.6.1.39),
an enzyme that participates in lysine biosynthesis and degradation, in some organisms.
This enzyme interconverts 2-aminoadipate and 2-oxoadipate, using alpha-ketoglutarate
as the amino acceptor. Gene candidates are found in
Homo sapiens (
Okuno et al., Enzyme Protein 47:136-148 (1993)) and
Thermus thermophilus (
Miyazaki et al., Microbiology 150:2327-2334 (2004)). The
Thermus thermophilus enzyme, encoded by
lysN, is active with several alternate substrates including oxaloacetate, 2-oxoisocaproate,
2-oxoisovalerate, and 2-oxo-3-methylvalerate.
Gene |
GenBank ID |
GI Number |
Organism |
aspC |
NP_415448.1 |
16128895 |
Escherichia coli |
AAT2 |
P23542.3 |
1703040 |
Saccharomyces cerevisiae |
ASP5 |
P46248.2 |
20532373 |
Arabidopsis thaliana |
got2 |
P00507 |
112987 |
Rattus norvegicus |
avtA |
YP_026231.1 |
49176374 |
Escherichia coli |
lysN |
BAC76939.1 |
31096548 |
Thermus thermophilus |
AadAT-II |
Q8N5Z0.2 |
46395904 |
Homo sapiens |
[0364] If the substrate is present in the D-stereoisomer, transamination can be catalyzed
by D-aminotransferase (EC 2.6.1.21), also known as D-amino acid aminotransferase and
D-alanine aminotransferase (DAAT). This class of enzyme is noted for its broad substrate
specificity, which is species-specific. The D-aminotransferase from
Bacillus species YM-1, encoded by
dat, has been cloned, sequenced (
Tanizawa et al., J Biol.Chem. 264:2450-2454 (1989)) and the crystal structure has been solved (
Peisach et al., Biochemistry 37:4958-4967 (1998)). This enzyme has also been engineered to alter the substrate specificity (
Gutierrez et al., Eur.J Biochem. 267:7218-7223 (2000);
Gutierrez et al., Protein Eng 11:53-58 (1998)). Additional gene candidates are found in
Bacillus licheniformis ATCC 10716 (
Taylor et al., Biochim.Biophys.Acta 1350:38-40 (1997)),
Staphylococcus haemolyticus (
Pucci et al., J Bacteriol. 177:336-342 (1995)) and
Bacillus subtilis (
Martinez-Carrion et al., J Biol.Chem. 240:3538-3546 (1965)).
Gene |
GenBank ID |
GI Number |
Organism |
dat |
P19938 |
118222 |
Bacillus sp. YM-1 |
dat |
P54692 |
1706292 |
Bacillus licheniformis ATCC 10716 |
dat |
P54694 |
1706294 |
Staphylococcus haemolyticus |
dat |
O0759.7.1 |
3121979 |
Bacillus subtilis |
[0365] The conversion of 2,4-diaminopentanoate to A-KP (Step N of Figure 13) is catalyzed
by an enzyme with 2,4-diaminopentanoate aminotrmisferase activity. Although this activity
is has not been characterized in enzymes to date, several enzymes catalyze a Similar
transformation, the conversion of 2,4-diaminobutanoate to aspartate-4-semialdehyde.
Exemplary enzyme candidates include beta-alanine aminotransferase (EC 2.6.1.18), diammobutyrate
aminotransferase (EC 2.6.1.46 and EC 2.6.1.76) and gamma-aminobutyrate (GABA) aminotransferase
(EC 2.6.1.19). An exemplary diaminobutyrate aminotransferase enzyme is encoded by
the
dat gene products in
Acinetobacter baumanii and
Haemophilus influenza (
Ikai et al., J Bacteriol. 179:5118-5125 (1997);
Ikai et al., Biol Pharm.Bull. 21:170-173 (1998)). In addition to its natural substrate, 2,4-diaminobutyrate, the
A.
baumanii DAT transaminates the terminal amines of lysine, 4-aminobutyrate and ornithine. Additional
diaminobutyrate aminotransferase gene candidates include the
ectB gene products of
Marinococcus halophilus and
Halobacillus dabanensis (Zhao et al., Curr Microbiol 53:183-188 (2006); Louis et al., Microbiology 143 (Pt 4):1141-1149 (1997)) and the
pvdH gene product of
Pseudomonas aeruginosa (
Vandenende et al., J Bacteriol. 186:5596-5602 (2004)). Diaminobutyrate aminotransferase enzymes that utilize alpha-ketoglutarate as an
amino acceptor are included in the EC class 2.6.1.76. Such enzymes are found in
Acinetobacter baumanii,
[0366] The beta-alanine aminotransferase of
Pseudomonas fluorescens also accepts 2,4-diammobutyrate as a substrate (
Hayaishi et al., J Biol Chem 236:781-790 (1961)); however, this activity has not been associated with a gene to date. Gamma-amiobutyrate
aminotransferase naturally interconverts succinic semialdehyde and glutamate to 4-aminobutyrate
and alpha-ketoglutarate. Generally, GABA aminotransferases react with a broad range
of alternate substrates (Schulz et al., 56:1-6 (1990); Liu et al., 43:10896-10905
(2004)). The two GABA transaminases in
E. coli are encoded by
gabT (
Bartsch et al., J Bacteriol. 172:7035-7042 (1990)) and
puuE (
Kurihara et al., J Biol.Chem. 280:4602-4608 (2005)). The
gabT gene product has been shown to have broad substrate specificity (Schutz et al., 56:1-6
(1990); Liu et at., 43:10896-10905 (2004)). GABA aminotransferases in
Mus musculus and
Sus scrofa have been shown to react with a range of alternate substrate (
Cooper, Methods Enzymol. 113:80-82 (1985)).
Gene |
GenBank ID |
GI Number |
Organism |
dat |
P56744.1 |
6685373 |
Acinetobacter baumanii |
dat |
P44951.1 |
1175339 |
Haemophilus influenzae |
ectB |
AABS7634.1 |
2098609 |
Marinococcus halophilus |
ectB |
AAZ57191.1 |
71979940 |
Halobacillus dabanensis |
pvdH |
AAG05801.1 |
9948457 |
Pseudomonas aeruginosa |
gabT |
NP_417148.1 |
16130576 |
Escherichia coli |
puuE |
NP_415818.1 |
16129263 |
Escherichia coli |
Abat |
NP_766549.2 |
37202121 |
Mus musculus |
gabT |
YP_257332.1 |
70733692 |
Pseudomonas fluorescens |
Abat |
NP_999428.1 |
47523600 |
Sus scrofa |
4.1.3.a Lyase: The condensation of pyruvate and acetaldehyde to 4-hydroxy-2-oxovalerate
(Step A of Figure 12) is catalyzed by 4-hydroxy-2-oxovalerate aldolase (EC 4.1.3.39).
This enzyme participates in pathways for the degradation of phenols, cresols and catechols.
The
E.
coli enzyme, encoded by
mhpE, is highly specific for acetaldehyde as an acceptor but accepts the alternate substrates
2-ketobutyrate or phenylpyruxate as donors (
Pollard et al., Appl Environ Microbiol 64:4093-4094 (1998)). Similar enzymes are encoded by the
cmtG and
todH genes of
Pseudomonas putida (
Lau et al., Gene 146:7-13 (1994);
Eaton, J Bacteriol. 178:1351-1362 (1996)). In
Pseudomonas CF600, this enzyme is part of a bifunctional aldolase-dehydrogenase heterodimer encoded
by
dmpFG (Manjasetty et al., Acta Crystallogr.D.Biol Crystallogr. 57:582-585 (2001)). The dehydrogenase functionality interconverts acetaldehyde and acetyl-CoA, providing
the advantage of reduced cellular concentrations of acetaldehyde, toxic to some cells.
Gene |
GenBank ID |
GI Number |
Organism |
mhpE |
AAC73455.1 |
1786548 |
Escherichia coli |
cmtG |
AAB62295.1 |
1263190 |
Pseudomonas putida |
todH |
AAA61944.1 |
485740 |
Pseudomonas putida |
dmpG |
CAA43227.1 |
45684 |
Pseudomonas sp. CF600 |
dmpF |
CAA43226.1 |
45683 |
Pseudomonas sp. CF600 |
[0367] 4.2.1.a. Dehydratase: Dehydration of 4-hydroxy-2-oxovalerate to 2-oxopentenoate (Step
B of Figure 12) is catalyzed by 4-hydroxy-2-oxovaterate hydratase (EC 4.2.1.80). A
similar enzyme is required to catalyze the dehydration of4-hydroxypent-2-enoate to
2,4-pentadienoate (Step D of Figure 13). 4-Hydroxy-2-oxovalerate hydratase participates
in aromatic degradation pathways and is typically co-transcribed with a gene encoding
an enzyme with 4-bydroxy-2-oxovaterate aldolase activity. Exemplary gene products
are encoded by
mhpD of
E. coli (
Ferrandez et al., J Bacteriol 179:2573-2581 (1997);
Pollard et al., Eur J Biochem. 251:98-106 (1998)),
todG and
cmtF of
Pseudomonas putida (
Lau et al., Gene 146:7-13 (1994);
Eaton, J Bacteriol. 178:1351-1362 (1996)),
cnbE of Comamonas sp. CNB-1 (
Ma et al., Appl Environ Microbiol 73:4477-4483 (2007)) and
mhpD of
Burkholderia xenovorans (
Wang et al., FEBS J 272:966-974 (2005)). A closely related enzyme, 2-oxohepta-4-ene-1,7-dioate hydratase, participates
in 4-hydroxyphenylacetic acid degradation, where it converts 2-oxo-hept-4-ene-1,7-dioate
(OHED) to 2-oxo-4-hydroxy-hepta-1,7-dioate using magnesium as a cofactor (
Burks et al., J.AmChem.Soc. 120: (1998)). OHED hydratase enzyme candidates have been identified and characterized in
E. coli C (
Roper et al., Gene 156:47-51 (1995);
Izumi et al., J Mol.Biol. 370:899-911 (2007)) and
E.
coli W (
Prieto et al., J Bacteriol. 178:111-120 (1996)). Sequence comparison reveals homologs in a wide range of bacteria, plants and animals.
Enzymes with highly similar sequences are contained in
Klebsiella pneumonia (91% identity, eval = 2e-138) and
Salmonella enterica (91% identity, eval = 4e-138), among others.
Gene |
GenBank ID |
GI Number |
Organism |
mhpD |
AAC73453.2 |
87081722 |
Escherichia coli |
cmtF |
AAB62293.1 |
1263188 |
Pseudomonas putida |
todG |
AAA61942.1 |
485738 |
Pseudomonas putida |
cnbE |
YP_001967714.1 |
190572008 |
Comamonas sp. CNB-1 |
mhpD |
Q13VU0 |
123358582 |
Burkho/deria xenovorans |
hpcG |
CAA57202.1 |
556840 |
Escheria coli C |
hpaH |
CAA86044.1 |
757830 |
Escherichia coli W |
hpaH |
ABR80130.1 |
150958100 |
Klebsiella pneumoniae |
Sari_01896 |
ABX21779.1 |
160865156 |
Salmonella enterica |
[0368] Enzyme candidates for catalyzing the dehydration of 2-hydroxypentenoate (Figure 12,
Step D) or 2-hydroxy-4-oxopentanoate (Figure 13, Step F) include fumarase (EC 4.2.1.2),
citramalate hydratase (EC 4.2.1.34) and dimethylmaleate hydratase (EC 4.2.1.85). Fumarase
enzymes naturally catalyze the reversible dehydration of malate to fumarate. Although
the ability of fumarase to react with 2-hydroxypentenoate or 2-hydroxy-4-oxopentanoate
as substrates has not been described in the literature, a wealth of structural information
is available for this enzyme and other researchers have successfully engineered the
enzyme to after activity, inhibition and localization (Weaver, 61:1395-1401 (2005)).
E. coli has three fumarases: FumA, FumB, and FumC that are regulated by growth conditions.
FumB is oxygen sensitive and only active under anaerobic conditions. FumA is active
under microanaerobic conditions, and FumC is the only active enzyme in aerobic growth
(Tseng et al., 183:461-467 (2001); Woods et al., 954:14-26 (1988);
Guest et al., J Gen Microbiol 131.2971-2984 (1985)). Additional enzyme candidates are found in
Campylobacter jejuni (
Smith et al., Int.J Biochem. Cell Biol 31:961-975 (1999)),
Thermus thermopolus (
Mizobata et al., Arch.Biochem.Biophys.355:49-55 (1998)) and
Rattus norvegicus (Kobayashi et al., 89:1923-1931 (1981)). Similar enzymes with high sequence homology
include
fum1 from
Arabidopsis thaliana and
fumC from
Corynebacterium glutamicum. The
mmcBC fumarase from
Pelotomaculum thermopropionicum is another class of fumarase with two subunits (Shimoyama et al., 270:207-213 (2007)).
Citramalate hydrolyase naturally dehydrates 2-methylmatate to mesaconate. This enzyme
has been studied in
Methanocaldococcus jannaschii in the context of the pyruvate pathway to 2-oxobutanoate, where it has been shown
to have a broad substrate specificity (
Drevland et al., J Bacteriol. 189:4391-4400 (2007)). This enzyme activity was also detected in
Colstridium tetanomorphum, Morganella morganii, Citrobacter amalonaticus where it is thought to participate in glutamate degradation (
Kato et al., Arch.Microbiol 168:457-463 (1997)). The
M.
jannaschii protein sequence does not bear significant homology to genes in these organisms.
Dimethylmaleate hydratase is a reversible Fe
2+-dependent and oxygen-sensitive enzyme in the aconitase family that hydrates dimethylmaeate
to form (2R,3S)-2,3-dimethytmalate. This enzyme is encoded by
dmdAB in
Eubacterium barkeri (Alhapel et al.,
supra;
Kollmann-Koch et al., Hoppe Seylers.Z.Physiol Chem. 365:847-857 (1984)).
Gene |
GenBank ID |
GI Number |
Organism |
fumA |
NP 416129.1 |
16129570 |
Escherichia coli |
fumB |
NP 418546.1 |
16131948 |
Escherichia coli |
fumC |
NP 416128.1 |
16129569 |
EScherichia coli |
fumC |
069294 |
9789756 |
Campylobacter jejuni |
fumC |
P84127 |
75427690 |
Thermus thremophilus |
fumH |
P14408 |
120605 |
Rattus norvegicus |
fumI |
P93033 |
39931311 |
Arcibidopsis thaliana |
fumC |
Q8NRN8 |
39931596 |
Corynebacterium glutamicum |
mmcB |
YP_001211906 |
147677691 |
Pelotomaculum thermopropionicum |
mmcC |
YP_001211907 |
147677692 |
Pelotomaculum thermopropionicum |
leuD |
Q58673.1 |
3122345 |
Methanocaldococcus jannashii |
dmdA |
ABC88408 |
86278276 |
Eubacterium barkeri |
dmdB |
ABC88409.1 |
86278277 |
Eubacterium barkeri |
[0369] 4.3.1 .a Ammonia-lyase: An ammonia lyase enzyme is required to catalyze the deaminatio
of 2-amino-4-oxopentanoate (AKP) to acetylacrylate in Step B of Figure 13. An enzyme
catalyzing this exact transformation has not been identified. However the AKP is structurally
similar to aspartate, the native substrate of aspartase (EC 4.3. 1.1.). Aspartase
is a widespread enzyme in microorganisms, and has been characterized extensively (Viola,
74:295-341 (2000)). The
E.
coli enzyme has been shown to react with a variety of alternate substrates including aspartatephenylmethyl
ester, asparagine, benzyl-aspartate and malate (Ma et al., 672:60-65 (1992)). In addition,
directed evolution was been employed on this enzyme to alter substrate specificity
(Asano et al., 22:95-101 (2005)). The crystal structure of the
E. coli aspartase, encoded by
aspA, has been solved (Shi et al., 36.9136-9144 (1997)). Enzymes with aspartase functionality
have also been characterized in
Haemophilus infuenzae (
Sjostrom et al., Biochim.Biophys.Acta 1324:182-190 (1997)),
Pseudomonas fluorescens (
Takagi et al.,.J.Bioehem. 96:545-552 (1984)),
Bacillus subtilis (Sjostrom et al., 1324:182-190 (1997)) and
Serratia marcescens (Takagi et al., 161:1-6(1985)).
Gene |
GenBank ID |
GI Number |
Organism |
aspA |
NP_418562 |
90111690 |
Escherichia coli K12 subsp. MG1655 |
aspA |
P44324.1 |
1168534 |
Haemophilus influenzae |
aspA |
P07346.1 |
114273 |
Pseudomonas fluorescens |
ansB |
P26899.1 |
251757243 |
Bacillus subtilis |
aspA |
P33109.1 |
416661 |
Serratia marcescens |
[0370] Another enzyme candidate for catalyzing the deamination of AKP is 3-methylaspartase
(EC 4.3.1.2). This enzyme, also known as beta-methylaspartase and 3-methylaspartate
ammonia-lyase, naturally catalyzes the deamination of threo-3-methylasparatate to
mesaconate. The 3-methylaspartase from
Clostridium, tetanomorphum has been cloned, functionally expressed in
E. coli, and crystallized (Asuncion et al., 57:731-733 (2001);
Asuncion et al., J Biol Chem. 277:8306-8311 (2002); Botting et al., 27:2953-2955 (1988); Goda et al., 31:10747-10756 (1992)). In
Citrobacter amalonaticus, this enzyme is encoded by
BAA28709 (
Kato and Asano, Arch.Microbiol 168:457-463 (1997)). 3-Methylaspartase has also been crystallized from
E. coli YG1002 (
Asano et al., FEMS Microbiol Lett. 118:255-258 (1994)) although the protein sequence is not listed in public databases such as GenBank.
Sequence homology can be used to identify additional candidate genes, including
CTC_02563 in
C. tetani and
ECs0761 in
Escherichia coli 0157:H7.
Gene |
Gen Bank ID |
GI Number |
Organism |
mal |
AAB24070.1. |
259429 |
Clostridium tetanomophorm |
BAA28709 |
BAA28709.1 |
3184397 |
Citrobacter amlolonaticus |
CTC_02563 |
NP_783085.1 |
28212141 |
Clostridium tetani |
ECs0761 |
BAB34184.1 |
13360220 |
Escherichia coli O157:H7 |
[0371] 5.1.1 .a Racemase: Racemase enzymes in the EC class 5.1.1 isomenze D- and L-amino
acids. Such an enzyme may be required to increase the bioavailability of D-alanine
and/or D-ornithine and thus enhance the conversion of alanine to AKP (Step A of Figure
13) or ornithine to 2,4-diaminopentanoate (Step M of Figure 13). Enzymes with alanine
racemase (EC 5.1.1.1) and ornithine racemase (EC 5.1.1.12) activity have been characterized.
Alanine racemase intereotiverts the L and D stereotsomers of alanine. Escherichia
coli has two alanine aminomutase enzymes, encoded
alr and
dadX (
Lilley et al., Gene 129:9-16 (1993);
Wild et al., Mol Gen Genet. 198:315-322 (1985)). The
vanT gene of
Entococcus gallinarum also exhibited alanine racemase activity when expressed in
E. coli (
Arias et al., Microbiology 146 (Pt 7): 1727-1734 (2000)). Additional alanine racemase enzyme candidates have been characterized in
Bacillus subtilis and
Mycrobacterium tuberculosis (
Pierce et al., FEMS Microbiol Lett. 283:69-74 (2008);
Strych et al., FEMS Microbiol Lett. 196:93-98 (2001)). Interconversion. of D-ornithine and L-ornithine is catalyzed by ornithine racemase.
The enzyme encoded by the
orr gene product of
C. sticklandii was purified and characterized (
Fonknechten, J.Bacteriol. In Press: (2009)). Additional ornithine racemase gene candidates can be identified by sequence similarity
in organisms such as
Clostridium difficile and
Fusobacterium periodonticum.
Gene |
GenBank ID |
GI Number |
Organism |
alr |
NP_418477.1 |
16131879 |
Escherichia coli |
dadX |
AAC74274.1 |
1787439 |
Escherichia coli |
vanT |
Q9X3P3.1 |
20140922 |
Enterococcus gallinarum |
yncD |
NP_389646.1 |
16078827 |
Bacillus subbtilis |
alr |
NP_338056.1 |
15843019 |
Mycobactrium tuberculosis CDC1551 |
alr |
NP_217940.1 |
15610559 |
Mycobacterium tuberculosis |
|
|
|
H37Rv |
orr |
CAQ42981.1 |
226885219 |
Clostridium sticklandii |
CdifA_020200002638 |
ZP_0S349631.1 |
255305459 |
Clostridium difficile |
FUSPEROL_00295 |
ZP-06025693.1 |
262066081 |
Fusobacterium periodonticum |
[0372] 5.4.3.a Aminomutase: Ornithine aminomutase (EC 5.4.3.5) catalyzes the conversion
of ornithine to 2,4-diaminopentanoate (Step M of Figure 13). A B1.2-dependent enzyme
with this activity, encoded by
oraSE of
Clostridium sticklandii, has been cloned, sequenced and expressed in
E. coli (
Chen et al., J.Biol.Chem. 276:44744-44750 (2001)). This enzyme preferentially reacts with the D-stereoisomer of ornithine (
Fonknechten, J.Bacteriol. In Press: (2009)). Ornithine aminomutase enzymes have not been characterized in other organisms to
date. Similar enzymes in organisms such as
Alkaliphilus oremlandii and
Clostridium difficile can be identified by sequence similarity. Lysine aminomutase catalyzes two similar
transformations: the interconversin of lysine with 2,5-diaminohexanoate (EC 5.4.3.4),
3,6-diaminohexanoate with 3,5-diaminohexalioate (EC 5.4.3.3). This enzyme participates
in the fermentation of lysine to acetate and butyrate and has been characterized in
Clostridium sticklandii (
Berkovitch et al., Proc.Ntal.Acad.Sci. U.S.A 101:15870-15875 (2004)) and
Porphyromonas gingivalis (
Tang et al., Biochemistry 41:8767-8776 (2002)).
Gene |
GenBank ID |
GI Number |
Organism |
oraE |
AAK72502.1 |
17223685 |
Clostridium sticklandii |
oraS |
AAK72501.1 |
17223684 |
C/ostridium sticklandii |
oraE (Clos_1695) |
YP_001513231.1 |
158320724 |
Alkaliphilus oremlandii |
oraS (Clos_1696) |
YP_001513232.1 |
158320725 |
Alkaliphilus oremlandii |
oraE |
ZP_05349629.1 |
255305457 |
Clostridium difficile |
oraS |
ZP_05349628.1 |
255305456 |
Clostridium difficile |
kamD |
AAC79717.1 |
3928904 |
Clostridium sticklandii |
kamE |
AAC79718.1 |
3928905 |
Clostridium sticklandii |
kamD |
NP_905288.1 |
34540809 |
Porphyromonas gingivalis W83 |
kamE |
NP_905289.1 |
34540810 |
Porphyromonas gingivalis W83 |
[0373] Other: 2-Amino-4-oxopentanoate (AKP) is formed from alanine and acetyl-CoA by AKP
thioloase (Step A in Figure 13). AKP thiolase (AKPT, no EC number) is a pyridoxal
phosphate-dependent enzyme that participates in omithine degradation in
Clostridium sticklandii (
Jeng et al., Biochemistry 13:2898-2903 (1974);
Kenklies et al., Microbiology 145 (Pt 4):819-826 (1999)). A gene cluster encoding the alpha and beta subunits of AKPT (
or-2
(ortA) and
or-3 (ortB)) was recently described and the biochemical properties of the enzyme were characterized
(
Fonknechten, J.Bacteriol In Press: (2009)). The enzyme is capable of operating in both directions and reacts with the D- isomer
of alanine. Enzyme engineering or directed evolution can enable the enzyme to function
with L-alanine as a substrate providing additional pathway versatility with regards
to the primary substrate. Alternately, co-expression of an alanine racemase enzyme
may enhance substrate availability. Enzymes with high sequence homology are found
in
Clostridium, difficile,
Alkaliphilus metalliredigenes QYF, Thermoanaerobacter sp. X514, and
Thermoanaerobacter tengcongensis MB4 (
Fonknechten, J.Bacteriol. In Press: (2009)).
Gene |
GenBank ID |
GI Number |
Organism |
ortA |
CAQ42979.1 |
226885215 |
Clostridium sticklandii |
ortB |
CAQ42980.1 GI: |
226885217 |
Clostridium sticklandii |
ortA |
YP_001086914.1 |
126698017 |
Clostridium difficile 630 |
ortB |
YP_001086915.1 |
126698018 |
Clostridium difficile 630 |
Amet_2368 |
YP_001320181.1 |
150390132 |
Alkaliphilus metalliredigenes QYF |
Amet_2369 |
YP_001320182.1 |
150390133 |
Alkaliphilus metalliredigenes QYF |
Teth514_1478 |
YP_001663101.1 |
167040116 |
Thermoanaerobacter sp. X514 |
Teth.514_1479) |
YP_001663102.1 |
167040117 |
Thermoanaerobacter sp. X514 |
TTE1235 |
NP_22858.1 |
20807687 |
Thermoanaerobacter tengcongensis MB4 |
thrC |
NP_622859.1 |
20807688 |
Thermoanaerobacter tengcongensis MB4 |
EXAMPLE IX
[0374] Exemplary Hydrogenase and CO Dehydrogenase Enzymes for Extracting Reducing Equivalents
from Syngav, and Exemplary Reductive TCA Cycle Enzymes
[0375] Enzymes of the reductive TCA cycle useful in the non-naturally occurring microbial
organisms of the present invention include one or more of ATP-citrate lyase and three
CO
2-fixing enzymes: isocitrate dehydrogenase, alpha-ketoglutarate:ferredoxin oxidoreductase,
pyruvate:ferredoxin oxidoreductase. The presence of ATP-citrate lyase or citrate lyase
and alpha-ketoglutarate:ferredoxin oxidoreductase, indicates the presence of an active
reductive TCA cycle in an organism. Enzymes for each step of the reductive TCA cycle
are shown below.
[0376] ATP-citrate lyase (ACL, EC 2.3.3.8), also called ATP citrate synthase, catalyzes
the ATP-dependent cleavage of citrate to oxaloacetate and acetyl-CoA. ACL is an enzyme
of the RTCA cycle that has been studied in green sulfur bacteria
Chlorobium limicola and
Clhlorobium tepidum. The alpha(4)beta(4) heteromeric enzyme from
Chlorobium limicola was cloned and characterized in
E. coli (
Kanao et al., Eur. J. Biochem. 269:3409-3416 (2002). The
C.
limicola enzyme, encoded by
aclAB, is irreversible and activity of the enzyme is regulated by the ratio of ADP/ATP.
A recombinant ACL from
Chlorobium tepidum was also expressed in
E.
coli and the holoenzyme was reconstituted in
vitro, in a study elucidating the role of the alpha and beta subunits in the catalytic mechanism
(
Kim and Tabita, J. Bacteriol. 188:6544-6552 (2006). ACL enzymes have also been identified in
Balnearium lithotrophicum, Sulfurihydrogenibium subterraneum and other members of the bacterial phylum
Aquifacae (
Hugler et al., Environ. Microbiol. 9:81-92 (2007)). This acitivy has been reported in some fungi as well. Exemplary organisms include
Sordaria macrospora (
Nowrousian et al., Curr. Genet. 37.189-93 (2000),
Aspergil/
us nidulans, Yarrowia lipolytica (
Hynes and Murray, Eukaryotic Cell, July: 1039-1048, (2010) and Aspergillus niger (Meijer et al. J. Ind. Microbiol. Biotechno/.36:1275-1280 (2009). Other candidates can be found based on sequence homology, Information related to
these enzymes is tabulated below:
Protein |
GenBank ID |
GI Number |
Organism |
aclA |
BAB21376.1 |
12407237 |
Ch/orobium limicola |
aclB |
BAB21375.1 |
12407235 |
Ch/orobium limicola |
aclA |
AAM72321.1 |
21647054 |
Ch/orobium tepidum |
aclB |
AAM72322.1 |
21647055 |
Chlorobium tepidum |
aclA |
ABI50076.1 |
114054981 |
Balnearium lithotrophicum |
aclB |
ABI50075.1 |
114054980 |
Balnearium lithotrophicum |
aclA |
ABI50085.1 |
114055040 |
Sulfurihydrogenibium subterraneum |
aclB |
ABI50084.1 |
114055039 |
Sulfurihydrogenibium subterraneum |
aclA |
AAX76834.1 |
62199504 |
Sulfurimonas denitrificans |
aclB |
AAX76835.1 |
62199506 |
Sulfurimonas denitrificans |
acl1 |
XP_504787.1 |
50554757 |
Yarrowia lipolytica |
acl2 |
XP_503231.1 |
50551515 |
Yarrowia lipolytica |
SPBC1703.07 |
NP_596202.1 |
19112994 |
Schizosaccharomyces pombe |
SPAC22A12.16 |
NP_593246.1 |
19114158 |
Schizosaccharomyces pombe |
acl1 |
CAB76165.1 |
7160185 |
Sordaria macrospora |
acl2 |
CAB76164.1 |
7160184 |
Sordaria macrospora |
aclA |
CBF86850.1 |
259487849 |
Aspergillus nidulans |
aclB |
CBF86848 |
259487848 |
Aspergillus nidulans |
[0377] In some organisms the conversion of citrate to oxaloacetate and acetyl-CoA proceeds
through a citryl-CoA intermediate and is catalyzed by two separate enzymes, citryl-CoA
synthetase (EC 6.2.1.18) and citryl-CoA lyase (EC 4.1.3.34) (
Aoshima, M., Appl. Microbiol. Biotechnol. 75:249-255 (2007). Citryl-CoA synthetase catalyzes the activation of citrate to citryl-CoA. The
Hydrogenobacter thermophilus enzyme is composed of large and small subunits encoded by
ccsA and
ccsB, respectively (
Aoshima et al., Mol. Micrbiol. 52:751-761 (2004)). The citryl-CoA synthetase of
Aquifex aeolicus is composed of alpha and beta subunits encoded by
sucC1 and
sucD1 (
Hugler et al., Environ. Microbiol. 9:81-92 (2007)). Citryl-CoA lyase splits citryl-CoA into oxaloacetate and acetyl-CoA. This enzyme
is a homotrimer encoded by
ccl in
Hy
drogenobacter thermophilus (
Aoshima et a]., Mol. Microbiol. 52:763-770 (2004)) and
aq_150 in
Aquifex aeolicus (Hugler et al.,
supra (2007)). The genes for this mechanism of converting citrate to oxaloacetate and citryl-CoA
have also been reported recently in
Chlorobium tepidum (
Eisen et al., PNAS 99(14): 9509-14 (2002).
Protein |
GenBank ID |
GI Number |
Organism |
ccsA |
BAD17844.1 |
46849514 |
Hydrogenobacter thermophilus |
ccsB |
BAD17846.1 |
46849517 |
Hydrogenobacter thermophilus |
sucC1 |
AAC07285 |
2983723 |
Aquifex aeolicus |
sucD1 |
AAC07686 |
2984152 |
Aquifex aeolicus |
ccl |
BAD17841.1 |
46849510 |
Hydrogenobacter thermophilus |
aq_150 |
AAC06486 |
2982866 |
Aquifex aeolicus |
CT0380 |
NP_661284 |
21673219 |
Chlorobium tepidum |
CT0269 |
NP_661173.1 |
21673108 |
Hydrobium tepidum |
CT1834 |
AAM73055.1 |
21647851 |
Chlorobium tepidum |
[0378] Oxaloacetate is converted into malate by malate dehydrogenase (EC 1.1.1.37), an enzyme
which functions in both the forward and reverse direction.
S.
cerevisiae possesses three copies of malate dehydrogenase,
MDH1 (
McAlister-Henn and Thompson, J. Bacteriol. 169:5157-5166 (1987),
MDH2 (
Minard and McAlister-Henn, Mol. Cell. Bio/. 11:370-380 (1991);
Gibson and McAlister-Henn, J. Biol. Chem. 278:25628-25636 (2003)), and
MDH3 (
Steffan and McAlister-Henn, J. Biol. Chem. 267:24708-24715 (1992)), which localize to the mitochondrion, cytosol, and peroxisome, respectively.
E.
coli is known to have an active malate dehydrogenase encoded by
mdh.
Protein |
GenBank ID |
GI Number |
Organism |
MDH1 |
NP_012838 |
6322765 |
Saccharomyces cerevisiae |
MDH2 |
NP_014515 |
116006499 |
Saccharomyces cerevisiae |
MDH3 |
NP_010205 |
6320125 |
Saccharomyces cerevisiae |
Mdh |
NP_417703.1 |
16131126 |
Escherichia coli |
[0379] Fumarate hydratase (EC 4.2.1.2) catalyzes the reversible hydration of fumarate to
malate. The three fumarases of
E.
coli, encoded by
fumA, fumB and
fumC, are regulated under different conditions of oxygen availability. FumB is oxygen
sensitive and is active under anaerobic conditions. FumA is active under microanaerobic
conditions, and FumC is active under aerobic growth conditions (
Tseng et al., J. Bacteriol. 183:461-467 (2001);
Woods et al., Biochim. Biophys. Acta 954:14-26 (1988);
Guest et al., J. Gen. Mcrobiol. 131:2971-2984 (1985)).
S.
cerevisiae contains one copy of a fumarase-encoding gene,
FUM1, whose product localizes to both the cytosol and mitochondrion (
Sass et al., J. Biol. Chem. 278:45109-45116 (2003)). Additional fumarase enzymes are found in
Campylobacter jejuni (
Smith et al., Int. J. Biochem. Cell. Biol. 31:961-975 (1999)),
Thermus thermophilus (
Mizobata et al., Arch. Biochem. Biophys. 355:49-55 (1998)) and
Rattus norvegicus (
Kobayashi et al., J, Biochem. 89:1923-1931 (1981)). Similar enzymes with high sequence homology include
fuml from
Arabidopsis thaliana and
fumC from
Corynebacterium glutamicum. The
MmcBC fumarase from
Pelotomaculum thermopropionicum is another class of fumarase with two subunits (
Shimoyama et al., FEMS Microbiol. Lett. 270:207-213 (2007)).
Protein |
GenBank ID |
GI Number |
Organism |
fumA |
NP_416129.1 |
16129570 |
Escherichia coli |
fumB |
NP_418546.1 |
16131948 |
Escherichia coli |
fumC |
NP_416128.1 |
16129569 |
Escherichia coli |
FUM1 |
NP_015061 |
6324993 |
Saccharomyces cerevisiae |
fumC |
Q8NRN8.1 |
39931596 |
Corynebacterium glutamicum |
Protein |
GenBank ID |
GI Number |
Organism |
fumC |
069294.1 |
9789756 |
Campylobacter jejuni |
fumC |
P84127 |
75427690 |
Thermus thermophilus |
fumH |
P14408.1 |
120605 |
Rattus norvegicus |
MmcB |
YP_001211906 |
147677691 |
Pelotomaculum thermopropionicum |
MmcC |
YP_001211907 |
147677692 |
Pelotomacu/um thermopropionicum |
[0380] Fumarate reductase catalyzes the reduction of fumarate to succinate. The fumarate
reductase of
E. coli, composed of four subunits encoded by
frdABCD, is membrane-bound and active under anaerobic conditions. The electron donor for
this reaction is menaquinone and the two protons produced in this reaction do not
contribute to the proton gradient (
Iverson et al., Science 284:1961-1966 (1999)). The yeast genome encodes two soluble fumarate reductase isozymes encoded by FRDS1
(
Enomoto et al., DNA Res. 3:263-267 (1996)) and FRDS2 (
Muratsubaki et al., Arch. Biochem. Biophys. 352:175-181 (1998)), which localize to the cytosol and promitochondrion, respectively, and are used
during anaerobic growth on glucose (
Arikawa et al., FEMS Microbiol. Lett. 165:111-116 (1998)).
Protein |
GenBank ID |
GI Number |
Organism |
FRDS1 |
P32614 |
418423 |
Saccharomyces cerevisiae |
FRDS2 |
NP_012585 |
6322511 |
Saccharomyces cerevisiae |
frdA |
NP_418578.1 |
16131979 |
Escherichia coli |
frdB |
NP_418577.1 |
16131978 |
Escherichia coli |
frdC |
NP_418576.1 |
16131977 |
Escherichia coli |
frdD |
NP_418475.1 |
16131877 |
Escherichia coli |
[0381] The ATP-dependent acylation of succinate to succinyl-CoA is catalyzed by succinyl-CoA
synthetase (EC 6.2.1.5). The product of the
LSC1 and
LSC2 genes of
S.
cerevisiae and the
sucC and
sucD genes of
E. coli naturally form a succinyl-CoA synthetase complex that catalyzes the formation of
succinyl-CoA from succinate with the concomitant consumption of one ATP, a reaction
which is reversible
in vivo (
Buck et al., Biochemistry 24:6245-6252 (1985)). These proteins are identified below:
Protein |
GenBank ID |
GI Number |
Organism |
LSC1 |
NP_014785 |
6324716 |
Saccharomyces cerevisiae |
LSC2 |
NP_011760 |
6321683 |
Saccharomyces cerevisiae |
sucC |
NP_415256.1 |
16128703 |
Escherichia coli |
sucD |
AAC73823.1 |
1786949 |
Escherichia coli |
[0382] Alpha-ketoglutarate:ferredoxin oxidoreductase (EC 1.2.7.3), also known as 2-oxoglutarate
synthase or 2-oxoglutarate:ferredoxin oxidoreductase (OFOR), forms alpha-ketoglutarate
from CO2 and succinyl-CoA with concurrent consumption of two reduced ferredoxin equivalents.
OFOR and pyruvate:ferredoxin oxidoreductase (PFOR) are members of a diverse family
of 2-oxoacid:ferredoxin (flavodoxin) oxidoreductases which utilize thiamine pyrophosphate,
CoA and iron-sulfur clusters as cofactors and ferredoxin, flavodoxin and FAD as electron
carriers
(Adams et al., Archaea. Adv. Protein Chem. 48:101-180 (1996)). Enzymes in this class are reversible and function in the carboxylation direction
in organisms that fix carbon by the RTCA cycle such as
Hydrogenobacter thermophilus, Desulfobacter hydrogenophilus and
Chlorobium species (Shiba et al. 1985;
Evans et al., Proc. Natl. Acad. ScI. U.S.A. 55:92934 (1966); Buchanan, 1971). The two-subunit enzyme from
H. thermophilus, encoded by
korAB, has been cloned and expressed in
E. coli (
Yun et al., Biochem. Biophys. Res. Commun. 282:589-594 (2001)). A five subunit OFOR from the same organism with strict substrate specificity for
succinyl-CoA, encoded by
forDABGE, was recently identified and expressed in
E.
coli (Yun et al. 2002). The kinetics of CO2 fixation of both
H. thermophilus OFOR enzymes have been characterized (
Yamamoto et al., Extremophiles 14:79-85 (2010)). A CO2-fixing OFOR from
Chlorobium thiosulfatophilum has been purified and characterized but the genes encoding this enzyme have not been
identified to date. Enzyme candidates in
Chlorobium species can be interred by sequence similarity to the
H. thermophilus genes. For example, the
Chlorobium limicola genome encodes two similar proteins. Acetogenic bacteria such as
Moorella thermoacetica are predicted to encode two OFOR enzymes. The enzyme encoded by
Moth_
0034 is predicted to function in the CO2-assimilating direction. The genes associated
with this enzyme,
Moth_0034 have not been experimentally validated to date but can be inferred by sequence
similarity to known OFOR enzymes.
[0383] OFOR enzymes that function in the decarboxylation direction under physiological conditions
can also catalyze the reverse reaction. The OFOR from the thermoacidophillic archaeon
Sulfolobus sp. strain 7, encoded by ST2300, has been extensively studied (Zhang et al. 1996.
A plasmid-based expression system has been developed for efficiently expressing this
protein in
E. coli (
Fukuda et al., Eur. J. Biochem. 268:5639-5646 (2001)) and residues involved in substrate specificity were determined (
Fukada and Wakagi, Biochim. Biophys. Acta 1597:74-80 (2002)). The OFOR encoded by
Ape1472/
Ape1473 from
Acropyrum pernix str. K1 was recently cloned into
E. coli, characterized, and found to react with 2-oxoglutarate and a broad range of 2-oxoacids
(
Nishizawa. et al., FEBS Lett. 579:2319-2322 (2005)). Another exemplary OFOR is encoded by
oorDABC in
Helicobacter pylori (Hughes et al. 1998). An enzyme specific to alpha-ketoglutarate has been reported
in
Thauera aromatica (
Dorner and Boll, J, Bactcriol, 184 (14), 3975-83 (2002). A similar enzyme can be found in
Rhodospillum rubrum by sequence homology. A two subunit enzyme has also been identified in
Chlorobium tepidum (
Eisen et al., PNAS 99(14): 9509-14 (2002)).
Protein |
GenBank ID |
GI Number |
Organism |
KorA |
BAB21494 |
12583691 |
Hydrogenobacter thermophilus |
KorB |
BAB21495 |
12583692 |
Hydrogenobacter thermophilus |
forD |
BAB62132.1 |
14970994 |
Hydrogenobacter thermophilus |
forA |
BAB62133.1 |
14970995 |
Hydrogenobacter thermophilus |
forB |
BAB62134.1 |
14970996 |
Hydrogenobacter thermophilus |
forG |
BAB62135.1 |
14970997 |
Hydrogenobacter thermophilus |
forE |
BAB62136.1 |
14970998 |
Hydrogenobacter thermophilus |
Clim_0204 |
ACD89303.1 |
189339900 |
Cholorobium limicola |
Clim_0205 |
ACD89302.1 |
189339899 |
Cholorobium limicola |
Clim_1123 |
ACD90192.1 |
189340789 |
Cholorobium limicola |
Clim_1124 |
ACD90193.1 |
189340790 |
Cholorobium limicola |
Moth_1984 |
YP_430825.1 |
83590816 |
Moorella thermoacetica |
Moth_1985 |
YP_430826.1 |
83590817 |
Moorella thermoacetica |
Moth_0034 |
YP_428917.1 |
83588908 |
Moorella thermoacetica |
ST2300 |
NP_378302.1 |
15922633 |
Sylfolobus sp. strain 7 |
Ape1472 |
BAA80470.1 |
5105156 |
Aeropyrum pernix |
Ape1473 |
BAA80471.2 |
116062794 |
Aeropyrum pernix |
oorD |
NP_207383.1 |
15645213 |
Helicobacter pylori |
oorA |
NP_207384.1 |
15645214 |
Helicobacter pylori |
oorB |
NP_207385.1 |
15645215 |
Helicobacter pylori |
oorC |
NP_207386.1 |
15645216 |
Helicobacter pylori |
CT0163 |
NP_661069.1 |
21673004 |
Cholorobium tepidum |
CT0162 |
NP_661068.1 |
21673003 |
Cholorobium tepidum |
korA |
CAA12243.2 |
19571179 |
Thauera aromatica |
korB |
CAD27440.1 |
19571178 |
Thauera aromatica |
Rru-A2721 |
YP_427805.1 |
83594053 |
Rhodospirillum rubrum |
Rru_A2722 |
YP_427806.1 |
83594054 |
Rhodospirillum rubrum |
[0384] Isocitrate dehydrogenase catalyzes the reversible decarboxylation of isocitrate to
2-oxoglutarate coupled to the reduction of NAD(P)
+. IDH enzymes in
Succharomyces cerevisiae and
Escherichia coli are encoded by
IDP1 and
icd, respectively (
Haselbeck and McAlister-Henn, J. Biol. Chem. 266:2339-2345 (1991);
Nimmo, H.G., Biochem. J. 234:317-2332 (1986)). The reverse reaction in the reductive TCA cycle, the reductive carboxylation of
2-oxoglutarate to isocitrate, is favored by the NADPH-dependent CO
2-fixing IDH from.
Chlorobium limicola and was fuctionally expressed in
E. coli (
Kanao et al., Eur. J. Biochem. 269:1926-1931 (2002)). A similar enzyme with 95% sequence identity is found in the
C. tepidum genome in addition to some other candidates listed below.
Protein |
GenBank ID |
GI Number |
Organism |
Icd |
ACI84720.1 |
209772816 |
Escherichia coli |
IDP1 |
AAA34703.1 |
171749 |
Saccharomyces cerevisiae |
Idh |
BAC00856.1 |
21396513 |
Chlorobium limicola |
Icd |
AAM71597.1 |
21646271 |
Chlorobium tepidum |
icd |
NP_952516.1 |
39996565 |
Geobacter sulfurreducens |
icd |
YP_393560. |
78777245 |
Sulfurimonas denitrificans |
[0385] In
H. thermophilus the reductive carboxylation of 2-oxoglutarate to isocitrate is catalyzed by two enzymes:
2-oxogtutarate carboxylase and oxatosuccinate reductase. 2-Oxogtutarate carboxylase
(EC 6.4.1.7) catalyzes the ATP-dependent carboxylation of alpha-ketoglutarate to oxalosuccinate
(
Aoshima and Igarashi, Mol. Microbiol. 62:748-759 (2006)). This enzyme is a large complex composed of two subunits. Biotinylation of the
large (A) subunit is required for enzyme function (
Aoshima et al., Mol. Microbiol. 51:-791-798 (2004)). Oxalosuccinate reductase (EC 1.1.1.-) catalyzes the NAD-dependent conversion of
oxatosuccinate to D-
threo-isocitrate. The enzyme is a homodimer encoded by
icd in
H. thermophilus. The kinetic parameters of this enzyme indicate that the enzyme only operates in the
reductive carboxylation direction
in vivo, in contrast to isocitrate dehydrogenases enzymes in other organisms (
Aoshima and tgarashi, J. Bacteriol. 190:2050-2055 (2008)). Based on sequence homology, gene candidates have also been found in
Thiobacillus denitrificans and
Thermocrinis albus.
Protein |
GenBank ID |
GI Number |
Organism |
cfiA |
BAF34932.1 |
116234991 |
Hydrogenobacter thermophilus |
cifB |
BAF34931.1 |
116234990 |
Hydrogenobacter thermophilus |
Icd |
BAD02487.1 |
38602676 |
Hydrogenobacter thermophilus |
Tbd_1556 |
YP_315314 |
74317574 |
Thiobacillus denitrificans |
Tbd_1555 |
YP_315313 |
74317573 |
Thiobacillus denitrificans |
Tbd_0854 |
YP_314612 |
74316872 |
Thiobacillus denitrificans |
Thal_0268 |
YP_003473030 |
289548042 |
Thermocrinis albus |
Thal_0267 |
YP_003473029 |
289548041 |
Thermocrinis albus |
Thal_0646 |
YP_003473406 |
289548418 |
Thermocrinis albus |
[0386] Aconitase (EC 4.2.1.3) is an iron-sulfur-containing protein catalyzing the reversible
isomerization of citrate and iso-citrate via the intermediate
cis-aconitate. Two aconitase enzymes are encoded in the
E. coli genome by
acnA and
acnB. AcnB is the main catabolic enzyme, while AcnA is more stable and appears to be active
under conditions of oxidative or acid stress (
Cunningham et al., Microbiology 143 (Pt 12):3795-3805 (1997)). Two isozymes of aconitase in
Salmonella typhimurium are encoded by
acnA and
acnB (
Horswill and Escalante-Semerena, Biochemisty 40:4703-4713 (2001)). The
S. cerevisiae aconitase, encoded by
ACO1, is localized to the mitochondria where it participates in the TCA cycle (
Gangloff et al., Mol. Cell. Biol. 10:3551-3561 (1990)) and the cytosol where it participates in the glyoxylate shunt (
Regev-Rudzki et al., Mol. Biol. Cell. 16:4163-4171 (2005)).
Protein |
GenBank ID |
GI Number |
Organism |
acnA |
AAC7438.1 |
1787531 |
Escherichia coli |
acnB |
AAC73229.1 |
2367097 |
Escherichia coli |
acnA |
NP_460671.1 |
16765056 |
Salmonella typhimurium |
HP0779 |
NP_207572.1 |
15645398 |
Helicobacter pylori 26695 |
H16_B0568 |
CAJ95365.1 |
113529018 |
Ralstonia eutropha |
DesfrDRAFT_3783 |
ZP_07335307.1 |
303249064 |
Desulfovibrio fructosovorans JJ |
Suden_1040 |
ABB44318.1 |
78497778 |
Sulfurimonas denitrificans |
(acnB) |
|
|
|
Hydth_0755 |
ADO45152.1 |
308751669 |
Hydrogenobacter thermophilus |
CT0543 (acn) |
AAM71785.1 |
21646475 |
Chlorobium tepidum |
Clim_2436 |
YP_001944436.1 |
189347907 |
Chlorobium limicola |
Clim_0515 |
ACD89607.1 |
189340204 |
Chlorobium limicola |
acnB |
NP_459163.1 |
16763548 |
Salmonella typhimurium |
ACO1 |
AAA34389.1 |
170982 |
Saccharomyces cerevisiae |
[0387] Pyruvate:ferredoxin oxidoreductase (PFOR) catalyzes the reversible oxidation of pyruvate
to form acetyl-CoA. The PFOR from
Desulfovibrio africanus has been cloned and expressed in
E. coli resulting in an active recombinant enzyme that was stable for several days in the
presence of oxygen (
Pieulle et al. J. Bacteriol. 179:5684-5692 (1997)). Oxygen stability is relatively uncommon in PFORs and is believed to be conferred
by a 60 residue extension in the polypeptide chain of the
D. africanus enzyme. Two cysteine residues in this enzyme form a disulfide bond that prtotects
it against inactivation in the form of oxygen. This disulfide bond and the stability
in the presence of oxygen, has been found in other
Desulfovibrio species also (
Vita et al., Biochemistry, 47: 957-64 (2008)). The
M. thermoacetica PFOR is also well characterized (
Menon and Pagsdale, Biochemistry 36:8484-8494 (1997)) and was shown to have high activity in the direction of pyruvate synthesis during
autotrophic growth (
Furdui and Ragsdale, J. Biol. Chem. 275:28494-28499 (2000)). Further,
E. coli possesses an uncharacterized open reading frame,
ydbK, encoding a protein that is 51% identical to the
M.
thermoacetica PFOR. Evidence for pyruvate oxidoreductase activity in
E. coli has been described (
Blaschkowski et al., Eur. J. Biochem, 123:563-569 (1982)). PFORs have also been described in other organisms, including
Rhodobacter capsulatas (
Yakunin and Hallenbeck, Biochimica et Biophysica Acta 1409 (1998) 39-49 (1998)) and
Choloboum tepidum (
Eisen et al., PNAS 99(14): 9509-14 (2002)). The five subunit PFOR from
H. thermophilus, encoded by
porEDABG, was cloned into
E. coli and shown to function in both the decarboxylating and CO
2-assimilating directions (Ikeda et al. 2006;
Yamamoto et al., Extremophiles 14:79-85 (2010)). Homologs also exist in
C. carboxidivorans P7. Several additional PFOR enzymes are described in the following
review (
Ragsdale, S.W., Chem. Rev. 103:2333-2346 (2003)). Finally, flavodoxin (e.g.,
fqrB from Helicobacter pylori or Campylobacter jejuni) (
St Maurice et al., J. Bacteriol. 189:4764-4773 (2007)) or Rnf-type proteins (
Seedorf et al., Proc. Natl. Acad. Sci. U.S.A. 105:2128-2133 (2008); and
Herrmann, J. Bacteriol 190:784-791 (2008)) provide a means to generate NADH or NADPH from the reduced ferredoxin generated
by PFOR. These proteins are identified below.
Protein |
GenBank ID |
GI Number |
Organism |
DesfrDRAFT_0121 |
ZP_07331646.1 |
303245362 |
Desulfovibrio fructosovorans JJ |
Por |
CAA70873.1 |
1770208 |
Desulfovibrio africanus |
por |
YP_012236.1 |
46581428 |
Desulfovibrio vulgaris str. Hildenborough |
Dde_3237 |
ABB40031.1 |
78220682 |
DesulfoVibrio desulfuricans G20 |
Ddes_0298 |
YP_002478891.1 |
220903579 |
Desulfovibrio desulfuricans subsp. desulfuricans str. ATCC 27774 |
Por |
YP_428946.1 |
83588937 |
Moorella thermoacetica |
YdbK |
NP_415896.1 |
16129339 |
Escherichia coli |
nifJ (CT1628) |
NP_662511.1 |
21674446 |
Chlorobium tepidum |
CJE1649 |
YP_179630.1 |
57238499 |
Campylobacter jejuni |
nifJ |
ADE85473.1 |
294476085 |
Rhodobacter capsulatus |
porE |
BAA95603.1 |
7768912 |
Hydrogenobacter thermophilus |
porD |
BAA95604.1 |
7768913 |
Hydrogenobacter thermophilus |
porA |
BAA95605.1 |
7768914 |
Hydrogenobacter thermophilus |
porB |
BAA95606.1 |
776891 |
Hydrogenobacter thermophilus |
porG |
BAA95607.1 |
7768916 |
Hydrogenobacter thermophilus |
FqrB |
YP_001482096.1 |
157414840 |
Campylobacter jejuni |
HP1164 |
NP_207955.1 |
15645778 |
Helicobacter pylori |
RnfC |
EDK33306.1 |
146346770 |
Clostridium kluyveri |
RnfD |
EDK33307.1 |
14646771 |
Clostridium kluyveri |
RnfG |
EDK33308.1 |
146446772 |
Clostridium kluyveri |
RnfE |
EDK33309.1 |
146346773 |
Clostridium kluyveri |
RnfA |
EDK33310.1 |
146346774 |
Clostridium kluyveri |
RnfB |
EDK33311.1 |
146346775 |
Clostridium kluyveri |
[0388] The conversion of pyruvate into acetyl-CoA can be catalyzed by several other enzymes
or their combinations thereof. For example, pyruvate dehydrogenase can transform pyruvate
into acetyl-CoA with the concomitant reduction of a molecule of NAD into NADH. It
is a multi-enzyme complex that catalyzes a series of partial reactions which results
in acylating oxidative decarboxylation of pyruvate. The enzyme comprises of three
submits: the pyruvate decarboxylase (E1), dihydrolipoamide acyltransferase (E2) dihydrolipoamide
dehydrogenase (E3). This enzyme is naturally present in several organisms, including
E. coli and
S.
cerevisiae. In the
E. coli. enzyme, specific residues in the E1 component are responsible for substrate specificity
(
Bisswanger, H., J. Biol. Chem. 256:815-82 (1981);
Bremer, J., Eur. J. Biochem. 8:535-540 (1969);
Gong et al., J. Biol. Chem. 275:13645-13653 (2000)). Enzyme engineering efforts have improved the
E. coli PDH enzyme activity under anaerobic conditions (
Kim et al., J. Bacteriol. 190:3851-3858 (2008);
Kim et al., Appl, Environ. Microbiol. 73:1766-1771 (2007);
Zhou et al., Biotcchnol. Lett. 30:335-342 (2008)). In contrast to the
E.
coli PDH, the
B. subtilis complex is active and for growth under anaerobic conditions (
Nakano et al., J. Bacteriol. 179:6749-6755 (1997)). The
Klelbsiella pneumoniae PDH, characterized during growth on glycerol, is also active under anaerobic conditions
(5). Crystal structures of the enzyme complex from bovine kidney (18) and the E2 catalytic
domain from
Azotobacter vinelandii are avaitable (4). Yet another enzyme that can catalyze this conversion is pyruvate
formate lyase. This enzyme catalyzes the conversion of pyruvate and CoA into acetyl-CoA
and formate. Pyruvate formate lyase is a common enzyme in prokaryotic organism that
is used to help modulate anaerobic redox balance. Exemplary enzymes can be found in
Escherichia coli encoded, by
pflB (
Knappe and Sawers, FEMS.Microbiol Rev. 6:383-398 (1990)),
Lactococcus lactis (
Melchiorsen et al., Appl Microbiol Biotechnot 58:338-344 (2002)), and
Streptococcus mutans (
Takahashi-Abbe et al, Oral.Microbiol Immunol. 18:293-297 (2003)).
E. coli possesses an additional pyruvate formate lyase, encoded by
tdcE. that catalyzes the conversion of pyruvate or 2-oxobutanoate to acetyl-CoA or propionyl-CoA,
respectively (
Hesslinger et al., Mol. Microbiol 27:477-492 (1998)). Both
pflB and
tclcE from
E. coli require the presence of pyruvate formate lyase activating enzyme, encoded by
pflA. Further, a short protein encoded by
yfiD in
E. coli can associate with and activity to oxygen-cleaved pyruvate formate lyase (
Vey et al., Proc.Natl. Acad. Sci. U.S.A. 105:16137-16141 (2008). Note that
pflA and
pflB from
E. coli were expressed in
S. cerevisiae as a means to increase cytosolic acetyt-CoA for butanol production as described in
WO/2008/080124]. Additional pyruvate formate lyase and activating enzyme candidates, encoded by
pfl and act, respectively, are found in
Clostridium pasteurianum (
Weidner et al., J Bacteriol. 178:2.440-2444 (1996)).
[0389] Further, different enzymes can be used in combination to convert pyruvate into acetyl-CoA.
For example, in
S. cerevisiae, acetyl-CoA is obtained in the cytosol by first decarboxylating pyruvate to form acetaldehyde;
the latter is oxidized to acetate by acetaldehyde dehydrogenase and subsequently activated
to form acetyt-CoA by acetyl-CoA synthetase. Acetyl-CoA synthetase is a native enzyme
in several other organisms including
E. coli (
Kumari et al., J. Bacteriol. 177:2878-2886 (1995)),
Salmonella enterica (
Starai et al., Microbiology 151:3793-3801 (2005);
Starai et al., J. Biol. Chem. 280:26200-26205 (2005)), and
Moorella thermoacetica (described already). Alternatively, acetate can be activated to form acetyl-CoA by
acetate kinase and phosphotransacetylase. Acetate kinase first converts acetate into
acetyl-phosphate with the accompanying use of an ATP molecule. Acetyl-phosphate and
CoA are next converted into acetyl-CoA with the release of one phosphate by phosphotransacetylase.
Both acetate kinase and phosphotransacetlyase are well-studied enzymes in several
Clostridia and
Methanosarcina thermophila.
[0390] Yet another way of converting pyruvate to acetyl-CoA is via pyruvate oxidase. Pyruvate
oxidase converts pyruvate into acetate, using ubiquione as the electron acceptor.
In
E. coli, this activity is encoded by
poxB.
PoxB has similarity to pyruvate decarboxylase of
S.
cerevisiae and
Zymomonas mobilis. The enzyme has a thiamin pyrophosphate cofactor (
Koland and Gennis, Biochemistry 21:4438-4442 (1982));
O'Brien et al., Biochemistry 16:3105-3109 (1977);
O'Brien and Gennis, J. Biol. Chem. 255:3302-3307 (1980)) and a flavin adenine dinucleotide (FAD) cofactor. Acetate can then be converted
into acetyl-CoA by either acetyl-CoA synthetase or by acetate kinase and phosphotransacetylase,
as described earlier. Some of these enzymes can also catalyze the reverse reaction
from acetyl-CoA to pyruvate.
[0391] For enzymes that use reducing equivalents in the form of NADH or NADPH, these reduced
carriers can be generated by transferring electrons from reduced ferredoxin. Two enzymes
catalyze the reversible transfer of electrons from reduced ferredoxins to NAD(P)
+, ferredoxin:NAD
+ oxidoreductase (EC 1.18.1.3) and ferredoxin:NADP
+ oxidoreductase (FNR, EC 1.18.1.2). Ferredoxin:NADP
+(FNR, EC 1.18.1.2) has a noncovalently bound FAD co factor that facilitates the reversible
transfer of electrons from NADPH to low-potential acceptors such as ferredoxins or
flavodoxins (
Blaschkowski et al., Eur. J. Biochem. 123:563-569(1992); Fujii et al., 1977). The
Helicobacter pylori FNR, encoded by
HP1164 (fqrB), is coupled to the activity of pyruvate:ferredoxin oxidoreductase (PFOR) resulting
in the pyruvate-dependent production of NADPH (St et al. 2007). An analogous enzyme
is found in
Campylobacter jejuni (St et al, 2007). A ferredoxin:NADP
+ oxidoreductase enzyme is encoded in the
E. coli genome by
fpr (Bianchi et al, 1993). Ferredoxin:NAD
+ oxidoreductase, utilizes reduced ferredoxin to generate NADH from NAD
+. In several organisms, including
E. coli, this enzyme is a component of multifunctional dioxygenase enzyme complexes. The ferredoxin:NAD
+ oxidoreductase of
E. coli, encoded by
hcaD, is a component of the 3-phenylproppionate dioxygenase system involved in involved
in aromatic acid utilization (Diaz et al. 1998). NADH:ferredoxin reductase activity
was detected in cell extracts of
Hydrogenobacter thermophilus strain TK-6, although a gene with this activity has not yet been indicated (Yoon
et al. 2006). Finally, the energy-conserving membrane-associated Rnf-type proteins
(
Seedorf et al., Proc. Natl. Acad. Sci. U.S.A. 105:2128-2133 (2008);
Herrmann et al., J. Bacteriol. 190:784-791 (2008)) provide a means to generate NADH or NADPH from reduced ferredoxin. Additional ferredoxin:NAD(P)+
oxidoreductases have been annotated in
Clostridium caboxydivorans P7.
Protein |
GenBank ID |
GI Number |
Organism |
HP1164 |
NP_207955.1 |
15645778 |
Helicobacter pylori |
RPA3954 |
CAE29395.1 |
39650872 |
Rhodopseudomonas palustris |
fpr |
BAH29712.1 |
225320633 |
Hydrogenobacter thermophilus |
yumC |
NP_391091.2 |
255767736 |
Bacillus subtilis |
CJE0663 |
AAW35824.1 |
57167045 |
Campylobacter jejuni |
fpr |
P28861.4 |
399486 |
Escherichia coli |
hcaD |
AAC75595.1 |
1788892 |
Escherichia coli |
LOC100282643 |
NP_001149023.1 |
226497434 |
Zea mays |
RnfC |
EDK33306.1 |
146346770 |
Clostridium kluyveri |
RnfD |
EDK33307.1 |
146346771 |
Clostridium kluyveri |
RnfG |
EDK3308.1 |
146346772 |
Clostridium kluyveri |
RnfE |
EDK33309.1 |
146346773 |
Clostridium kluyveri |
RnfA |
EDK33310.1 |
146346774 |
Clostridium kluyveri |
RnfB |
EDK33311.1 |
146346775 |
Clostridium kluyveri |
CcarbDRAFT_2639 |
ZP_05392639.1 |
255525707 |
Clostridium carboxidivorans P7 |
CcarbDRAFT_2638 |
ZP_05392638.1 |
255525706 |
Clostridium carboxidivorans P7 |
CcarbDRAFT_2636 |
ZP_05392636.1 |
255525704 |
Clostridium carboxidivorans P7 |
CcarbDRAFT_5060 |
ZP_05395060.1 |
255528241 |
Clostridium carboxidivorans P7 |
CcarbDRAFT_2450 |
ZP_05392450.1 |
255525514 |
Clostridium carboxidivorans P7 |
CcarbDRAFT_1084 |
ZP_05391084.1 |
255524124 |
Clostridium carboxidivorans P7 |
[0392] Ferredoxins are small acidic proteins containing one or more iron-sulfur clusters
that function as intracellular electron carriers with a low reduction potential. Reduced
ferredoxins donate electrons to Fe-dependent enzymes such as ferredoxin-NADP
+ oxidoreductase, pyruvate:ferredoxin oxidoreductase (PFOR) and 2-oxoglutarate:ferredoxin
oxidoreductase (OFOR). The
H. thermophilus gene
fdx1 encodes a [4Fe-4S]-type ferredoxin that is required for the reversible carboxylation
of 2-oxoglutarate and pyruvate by OFOR and PFOR, respectively (
Yamamoto et al., Extremophiles 14:79-85 (2010)). The ferredoxin associated with the
Sulfolobus solfataricus 2-oxoacid:ferredoxin reductase is a monomeric dicluster [3Fe-4S][4Fe-4S] type ferredoxin
(Park et al. 2006). While the gene associated with this protein has not been fully
sequenced, the N-terminal domain shares 93% homology with the
zfx ferredoxin from
S. acidocaldarius. The
E. coli genome encodes a soluble ferredoxin of unknown physiological function,
fdx. Some evidence indicates that this protein can function in iron-sulfur cluster assembly
(Takahashi and Nakamura, 1999). Additional ferredoxin proteins have been characterized
in
Helicobacterer pylori (Mukhopadhyay et al. 2003) and
Campylobacter jejuni (van Vliet et al. 2001). A 2Fe-2S ferredoxin from
Clostridium pasteurianum has been cloned and expressed in
E. coli (
Fujinaga and Meyer, Biochemical and Biophysical Research Communications, 192(3): (1993)). Acetogenic bacteria such as
Moorella thermoacetica, Clostridium carboxidivorans P7 and Rhodospirillum rubrum are predicted to encode several ferredoxins, listed in the table below.
Protein |
GenBank ID |
GI Number |
Organism |
fdxl |
BAE02673.1 |
68163284 |
Hydrogenobacter thermophilus |
M11214.1 |
AAA83524.1 |
144806 |
Clostridium Moorella |
Zfx |
AAY79867.1 |
68566938 |
Sulfolobus acidocalarius |
Fdx |
AAC75578.1 |
1788874 |
Escherichia coli |
hp_0277 |
AAD07340.1 |
2313367 |
Helicobacter pylori |
fdxA |
CAL34484.1 |
112359698 |
Campylobacter jejuni |
Moth_0061 |
ABC18400.1 |
83571848 |
Moorella thermoacetica |
Moth_1200 |
ABC19514.1 |
83572962 |
Moorella thermoacetica |
Moth_1888 |
ABC20188.1 |
83573636 |
Moorella thermoacetica |
Moth_2112 |
ABC20404.1 |
83573852 |
Moorella thermoacetica |
Moth_1037 |
ABC19351.1 |
83572799 |
Moorella thermoacetica |
CcarbDRAFT_4383 |
ZP_05394383.1 |
255527515 |
Clostridium carboxidivorans P7 |
CcarbDRAFT_2958 |
ZP_05392958.1 |
255526034 |
Clostridium carboxidivorans P7 |
CcarbDRAfT_2281 |
ZP_05392281.1 |
255525342 |
Clostridium carboxidivorans P7 |
CcarbDRAFT_5296 |
ZP_05395295.1 |
255528511 |
Clostridium carboxidivorans P7 |
CcarbDRAFT_1615 |
ZP_05391615.1 |
255524662 |
Clostridium carboxidivorans P7 |
CCarbDRAFT_1304 |
ZP_05391304.1 |
255524347 |
Clostridium carboxidivorans P7 |
cooF |
AAG29808.1 |
11095245 |
Carboxydothermus hydrogenoformans |
fdxN |
CAA35699.1 |
46143 |
Rhodobacter capsulatus |
Rru_A2264 |
ABC23064.1 |
83576513 |
Rhodospirillum rubrum |
Rru_A1916 |
ABC22716.1 |
83576165 |
Rhodospirillum rubrum |
Rru_A2026 |
ABC22826.1 |
83576275 |
Rhodospirillum rubrum |
cooF |
AAC45122.1 |
1498747 |
Rhodospirillum rubrum |
fdxN |
AAA26460.1 |
152605 |
Rhodospirillum rubrum |
Alvin_2884 |
ADC63789.1 |
288897953 |
Allochromatium vinosum DSM 180 |
fdx |
YP_002801146.1 |
226946073 |
Azotobacter vinelandii DJ |
CKL_3790 |
YP_001397146.1 |
153956381 |
Clostridium kluyveri DSM 555 |
fer1 |
NP_949965.1 |
39937689 |
Rhodopseudomonas palustris CGA009 |
fdx |
CAA12251.1 |
3724172 |
Thaucra aromatica |
CHY_2405 |
YP_361202.1 |
78044690 |
Carboxydothermus hydrogenoformans |
|
fer YP_359966.1 |
|
Carboxydothermus hydrogenoformans |
fer |
AAC83945.1 |
1146198 |
Bacillus subtilis |
fdx1 |
NP_249053.1 |
15595559 |
Pseudomonas aeruginosa PA01 |
yfhL |
AP_003148.1 |
89109368 |
Escherichia coli K-12 |
[0393] Succinyl-CoA transferase catalyzes the conversion of succinyl-CoA to succinate while
transferring the CoA moiety to a CoA acceptor molecule. Many transferases have broad
specificity and can utilize CoA acceptors as diverse as acetate, succinate, propionate,
butyrate, 2-methylacetoacetate, 3-ketohexanoate, 3-ketopentanoate, valerate, crotonate,
3-mercaptopropionate, propionate, vinylacetate, and butyrate, among others,
[0394] The conversion of succinate to succinyl-CoA can be carried by a transferase which
does not require the direct consumption of an ATP or GTP. This type of reaction is
common in a number of organisms. The conversion of succinate to succinyl-CoA can also
be catalyzed by succinyl-CoA:Acetyl-CoA transferase. The gene product of
cat1 of
Clostridium kluyveri has been shown to exhibit succinyl-CoA: acetyl-CoA transferase activity (
Sohling and Gottschalk, J. Bacteriol. 178:871-880 (1996)). In addition, the activity is present in
Trichomonas vaginalis (van Grinsven et al. 2008) and
Trypanosoma brucei (Riviere et al. 2004). The succinyl-CoA:acetate CoA-transferase from
Acetobacter aceti, encoded by
aarC, replaces succinyl-CoA synthetase in a variant TCA cycle (Mullins et al. 2008). Similar
succinyl-CoA transferase activities are also present in
Trichomonas vaginalis (van Grinsven et al. 2008),
Trypanosoma brucei (Riviere et al. 2004) and
Clostridium kluveri (Sohling and Gottschalk, 1996c). The beta-ketoadipate:succinyl-CoA transferase encoded
by
pcaI and
pcaJ in
Pseudomonas putida is yet another candidate (Kaschabek et al. 2002). The aforementioned proteins are
identified below.
Protein |
GenBank ID |
GI Number |
Organism |
cat1 |
P38946.1 |
729048 |
Clostridium kluyveri |
TVAG_395550 |
XP_001330176 |
123975034 |
Trichomonas vaginalis G3 |
Tb11.02.0290 |
XP_828352 |
71754875 |
Trypanosoma brucei |
pcaI |
AAN69545.1 |
24985644 |
Pseudomonas putida |
pcaJ |
NP_746082.1 |
26990657 |
Pseudomonas putida |
aarC |
ACD85596.1 |
189233555 |
Acetobacter aceti |
[0395] An additional exemplary transferase that converts succinate to succinyl-CoA while
converting a 3-ketoacyl-CoA to a 3-ketoacid is succinyl-CoA:3:ketoacid-CoA transferase
(EC 2.8.3.5). Exemplary succinyl-CoA:3-ketoacid-CoA transferases are present in
Helicobacter pylori (Corthesy-Theulaz et al. 1997),
Bacillus subtilis, and
Homo sapiens (Fukao et al. 2000; Tanaka et al. 2002). The aforementioned proteins are identified
below.
Protein |
GenBank ID |
GI Number |
Organism |
HPAG1_0676 |
YP_627417 |
108563101 |
Helicobacter pylori |
HPAG1_0677 |
YP_627418 |
108563102 |
Helicobacter pylori |
|
ScoA |
NP_391778 |
16080950 |
Bacillus subtilis |
|
ScoB |
NP_391777 |
16080949 |
Bacillus subtilis |
|
OXCT1 |
NP_000427 |
4557817 |
Homo sapiens |
|
OXCT2 |
NP_071403 |
11545841 |
Homo sapiens |
[0396] Converting succinate to succinyl-CoA by succinyl-CoA:3:ketoacid-CoA transferase requires
the simultaneous conversion of a 3-ketoacyl-CoA such as acetoacetyl-CoA to a 3-ketoacid
such as acetoacetate. Conversion of a 3-ketoacid back to a 3-ketoacyl-CoA can be catalyzed
by an acetoacetyl-CoA:acetate:CoA transferase. Acetoacetyl-CoA:acetate:CoA transferase
converts acetoacetyl-CoA and acetate to acetoacetate and acetyl-CoA, or vice versa.
Exemplary enzymes include the gene products of
atoAD from
E. coli (
Hanai et al., Appl Environ Microbiol 73:7814-7818 (2007),
ctfAB from
C. acetobutylicum (
Jojima et al., Appl Microbiol Biotechnol 77:1219-1224 (2008), and
ctfAB from
Clostridium saccharoperbutylacetonicum (
Kosaka et al., Biosci.Biotechnol Biochem. 71:58-68 (2007)) are shown below.
Protein |
GenBank ID |
GI Number |
Organism |
AtoA |
NP_416726.1 |
2492994 |
Escherichia coli |
AtoD |
NP_416725.1 |
2492990 |
Escherichia coli |
CtfA |
NP_149326.1 |
15004866 |
Clostridium acetobutylicum |
CtfB |
NP_149327.1 |
15004867 |
Clostridium acetobutylicum |
CtfA |
AAP42564.1 |
31075384 |
Clostridium saccharoperbutylacetonicum |
CtfB |
AAP42565.1 |
31075385 |
Clostridium saccharoperbutylacetonicum |
[0397] Yet another possible CoA acceptor is benzylsuccinate. Succinyl-CoA:(R)-Benzylsuccinate
CoA-Transferase functions as part of an anaerobic degradation pathway for toluene
in organisms such as
Thauera aromatica (
Leutwein and Heider, J. Bact. 183(14) 4288-4295 (2001)). Homologs can be found in
Azoarcus sp. T,
Aromatoleum aromaticum EbN1, and
Geobacter metallireducens GS-15. The aforementioned proteins are identified below.
Protein |
GenBank ID |
GI Number |
Organism |
bbsE |
AAF89840 |
9622535 |
Thauera aromatic |
Bbsf |
AAF89841 |
9622536 |
Thauera aromatic |
bbsE |
AAU45405.1 |
52421824 |
Azoarcus sp. T |
bbsF |
AAU45406.1 |
52421825 |
Azoarcus sp. T |
bbsE |
YP_158075.1 |
56476486 |
Aromatoleum aromaticum EbN1 |
bbsF |
YP_158074.1 |
56476485 |
Aromatoleum aromaticum EbN1 |
Gmet_1521 |
YP_384480.1 |
78222733 |
Geobacter metallireducens GS-15 |
Gmet_1522 |
YP_384481.1 |
78222734 |
Geobacter metallireducens GS-15 |
[0398] Addittionally,
ygfH encodes a propionyl CoA:succinate CoA transferase in
E. coli (
Haller et al., Biochemistry, 39(16) 4622-4629). Close homologs can be found in, for example,
Citrobacter youngae ATCC 29220,
Salmonella enterica subsp.
arizonae serovar, and
Yersinia intermedia ATCC 29909. The aforementioned proteins are identified below.
Protein |
GenBank ID |
GI Number |
Organism |
ygfH |
NP_417395.1 |
16130821 |
Escherichia coli str. K-12 substr. MG1655 |
CIT292_04485 |
ZP_03838384.1 |
227334728 |
Citrobacter youngae ATCC 29220 |
SARI_04582 |
YP_001573497.1 |
161506385 |
Salmonella enterica subsp. arizonae serovar |
yinte0001_14430 |
ZP_04635364.1 |
238791727 |
Yersinia intermedia ATCC 29909 |
[0399] Citrate lyase (EC 4.1.3.6) catalyzes a series of reactions resulting in the cleavage
of citrate to acetate and oxaloacetate. The enzyme is active under anaerobic conditions
and is composed of three subunits: an acyl-carrier protein (ACP, gamma), an ACP transferase
(alpha), and a acyl lyase (beta). Enzyme activation uses covalent binding and acetylation
of an unusual prosthetic group, 2'-(5"-phosphoribosyl)-3-'-dephospho-CoA, which is
similar in structure to acetyl-CoA. Acylation is catalyzed by CitC, a citrate lyase
synthetase. Two additional proteins, CitG and CitX, are used to convert the apo enzyme
into the active holo enzyme (
Schneider et al., Biochemistry 39:9438-9450 (2000)). Wild type
E. coli does not have citrate lyase activity; however, mutants deficient in molybdenum cofactor
synthesis have an active citrate lyase (
Clark, FEMS Microbiol. Lett. 55:245-249 (1990)). The
E. coli enzyme is encoded by
citEFD and the citrate lyase synthetase is encoded by
citC (
Nilekani and SivaRaman, Biochemistry 22:4657-4663 (1983)). The
Leuconostoc mesenteroides citrate lyase has been cloned, characterized and expressed in
E. coli (
Bekal et al., J. Bacteriol. 180:647-654 (1998)). Citrate lyase enzymes have also been identified in enterobacteria that utilize
citrate as a carbon and energy source, including
Salmonella typhimurium and
Klebsiella pneumoniae (
Bott, Arch. Microbiol. 167: 78-88 (1997);
Bott and Dimroth, Mol. Microbiol. 14:347-356 (1994)). The aforementioned proteins are tabulated below.
Protein |
GenBank ID |
GI Number |
Organism |
citF |
AAC73716.1 |
1786832 |
Escherichia coli |
Cite |
AAC73717.2 |
87081764 |
Escherichia coli |
citD |
AAC73718.1 |
1786834 |
Escherichia coli |
citC |
AAC73719.2 |
87081765 |
Escherichia coli |
citG |
AAC73714.1 |
1786830 |
Escherichia coli |
citX |
AAC73715.1 |
1786831 |
Escherichia coli |
citF |
CAA71633.1 |
2842397 |
Leuconostoc mesenteroides |
Cite |
CAA71632.1 |
2842396 |
Leuconostoc mesenteroides |
citD |
CAA71635.1 |
2842395 |
Leuconostoc mesenteroides |
citC |
CAA71636.1 |
3413797 |
Leuconostoc mesenteroides |
citG |
CAA71634.1 |
2842398 |
Leuconostoc mesenteroides |
citX |
CAA71634.1 |
2842398 |
Leuconostoc mesenteroides |
citF |
NP_459613.1 |
16763998 |
Salmonella typhimurium |
cite |
AAL19573.1 |
16419133 |
Salmonella typhimurium |
citD |
NP_459064.1 |
16763449 |
Salmonella typhimurium |
citC |
NP_459616.1 |
16764001 |
Salmonella typhimurium |
citG |
NP_459611.1 |
16763996 |
Salmonella typhimurium |
citX |
NP_459612.1 |
16763997 |
Salmonella typhimurium |
citF |
CAA56217.1 |
565619 |
Klebsiella pneumoniae |
cite |
CAA56216.1 |
565618 |
Klebsiella pneumoniae |
citD |
CAA56215.1 |
565617 |
Klebsiella pneumoniae |
citC |
BAH66541.1 |
238774045 |
Klebsiella pneumoniae |
citG |
CAA56218.1 |
565620 |
Klebsiella pneumoniae |
citX |
AAL60463.1 |
18140907 |
Klebsiella pneumoniae |
[0400] Acetate kinase (EC 2.7.2.1) catalyzes the reversible ATP-dependent phosphorylation
of to acetytphosphate. Exemplary acetate kinase enzymes have been characterized in
many organisms including
E.
coli, Clostridium acetobutylicum and
Methanosarcina thermophila (
Ingram-Smith et at., J. Bacteriol. 187:2386-2394 (2005);
Fox and Roseman, J. Biol. Chem. 261:13487-13497 (1986);
Winzer et al., Microbioloy 143 (Pt 10):3279-3286 (1997)), Acetate kinase activity has also been demonstrated in the gene product of
E.
coli purT (
Marolewski et al., Biochemistry 33:2531-2537 (1994). Some butyrate kinase enzymes (EC 2.7.2.7), for example
buk1 and
buk2 from
Clostridium acetobutylicum, also accept acetate as a substrate (
Hartmanis, M.G., J. Biol. Chem. 262:617-621 (1987)).
Protein |
GenBank ID |
GI Number |
Organism |
ackA |
NP_416799.1 |
16130231 |
Escherichia coli |
Ack |
AAB18301.1 |
1491790 |
Clostridium acetobutylicum |
Ack |
AAA72042.1 |
349834 |
Methanosarcina thermophila |
purT |
AAC74919.1 |
1788155 |
Escherichia coli |
buk1 |
NP_349675 |
15896326 |
Clostridium acetobutylicum |
buk2 |
Q97II1 |
20137415 |
Clostridium acetobutylicum |
[0401] The formation of acetyl-CoA from acetylphosphate is catalyzed by phosphotransacetylase
(EC 2.3.1.8). The
pta gene from
E.
coli encodes an enzyme that reversibly converts acetyl-CoA into acetyl-phosphate (
Suzuki, T., Biochim. Biophys. Acta 191 :559-569 (969)). Additional acetyltransferase enzymes have been characterized in
Bacillus subtilis (
Rado and Hoch, Biochim. Biophys. Acta 321:114-125 (1973),
Clostridium kluyveri (
Stadtman, E., Methods Enzymol. 1:5896-599 (1955), and
Thermotoga maritima (
Bock et al., J. Bacteriol. 181:1861-1867 (1999)). This reaction is also catalyzed by some phosphotranbutyrylase enzymes (EC 2.3.1.19)
including the
ptb gene products from
Clostridium acetobutylicum (
Wiesenborn et al., App. Environ, Microbiol. 55.317-322 (1989);
Welter et al., Gene 134:107-111 (1993)). Additional
ptb genes are found in butyrate-producing bacterium L2-50 (
Louis et al., J. Bacteriol. 186:2099-2106 (2004) and
Bacillus megaterium (
Vazquez et al., Curr. Microbiol. 42:345-349 (2001).
Protein |
GenBank ID |
CI Number |
Organism |
Pta |
NP_416800.1 |
71152910 |
Escherichia coli |
Pta |
P39646 |
730415 |
Bacillus subtilis |
Pta |
A5N801 |
146346896 |
Clostridium kluyveri |
Pta |
Q9X0LA |
6685776 |
Thermotoga maritima |
Ptb |
NP_349676 |
34540484 |
Clostridium acetobutylicum |
Ptb |
AAR19757.1 |
38425288 |
butyrate-producing bacterium L2-50 |
Ptb |
CAC07932.1 |
10046659 |
Bacillus megaterium, |
[0402] The acylation of acetate to acetyl-CoA is catalyzed by enzymes with acetyl-CoA synthetase
activity. Two enzymes that catalyze this reaction are AMP-forming acetyl-CoA synthetase
(EC 6.2.1. 1) and ADP-forming acetyl-CoA synthetase (EC 6.2.1.13). AMP-forming acetyl-CoA
synthetase (ACS) is the predominant enzyme for activation of acetate to acetyl-CoA.
Exemplary ACS enzymes are found in
E. coli (
Brown et al., J. Gen. Microbiol. 102:327-336 (1977)),
Ralstonia eutropha (
Priefert and Steinbuchel, J. Bacteriol. 174:6590-6599 (1992)),
Methanothermobacter thermautotrophicus (
Ingram-Smith and Smith, Archaea 2:95-107 (2007)),
Slamonella enterica (
Gulick et al., Biochemistry 42:2866-2873 (2003)) and
Saccharomyces cerevisiae (
Jogl and Tong, Biochemistry 43:1425-1431 (2004)). ADP-forming acetyl-CoA synthetases are reversible enzymes with a generally broad
substrate range (
Musfeldt and Schonheit, J. Bacteriol. 184:636-644 (2002)). Two isozymes of ADP-forming acetyl-CoA synthetases are encoded in the
Archaeoglobus fulgidus genome by are encoded by AF1211 and AF1983 (Musfeldt and Schonheit,
supra (2002)). The enzyme from
Haloarcula marismortui (annotated as a succinyl-CoA synthetase) also accepts acetate as a substrate and
reversibility of the enzyme was demonstrated (
Brasen and Schonheit, Arch. Microbiol. 182:277-287 (2004)). The ACD encoded by
PAE3250 from hyperthermophilic crenarchaeon
Pyrobaculum aerophilum showed the broadest substrate range of all characterized ACDs, reacting with acetate,
isobutyryl-CoA (preferred substrate) and phenylacetyl-CoA (Brasen and Schonheit,
supra (2004)). Directed evolution or engineering can be used to modify this enzyme to operate
at the physiological temperature of the host organism. The enzymes from
A. fulgidus, H. marismortui and
P.
aerophilum have all been cloned, functionally and characterized in
E.
coli (Brasen and Schonheit,
supra (2004); Musfeldt and Schonheit,
supra (2002)). Additional candidates include the succinyl-CoA synthetase encoded by
sucCD in
E. coli (
Buck et al., Biochemistry 24:6245-6252 (1985)) and the acyl-CoA ligase from
Pseudomonas putida (
Fernandez-Valverde et al., Appl. Environ. Microbiol. 59.1149-1154 (1993)). The aforementioned proteins are tabulated below.
Protein |
GenBank ID |
GI Number |
Organism |
acs |
AAC77039.1 |
1790505 |
Escherichia coli |
acoE |
AAA21945.1 |
141890 |
Ralstonia eutropha |
acs1 |
ABC87079.1 |
86169671 |
Methanothermobacter thermautotrophicus |
acs1 |
AAL23099.1 |
16422835 |
Salmonella enterica |
ACS1 |
Q01574.2 |
257050994 |
Saccharomyces cerevisiae |
AF1211 |
NP_070039.1 |
11498810 |
Archaeoglobus fulgidus |
AF1983 |
NP_070807.1 |
11499565 |
Archaeoglobus fulgidus |
scs |
YP_135572.1 |
55377722 |
Haloarcula marismortui |
PAE3250 |
NP_560604.1 |
18313937 |
Pyrobaculum aerophilum str. IM2 |
sucC |
NP_415256.1 |
16128703 |
Escherichia coli |
sucD |
AAC73823.1 |
1786949 |
Escherichia coli |
paaF |
AAC24333.2 |
22711873 |
Pseudomonas putida |
[0403] Conversion of acetyl-CoA to malonyl-CoA can be carried out by an acetyl-CoA carboxylase
enzyme. These enzymes contain multiple subunits. Three of such enzymes are provided
below.
Gene |
Organism |
Accession number |
GI Number |
accA |
Escherichia coli K-12 |
AAC73296.1 |
1786382 |
accB |
Escherichia coli K-12 |
AAC76287.1 |
1789653 |
accC |
Escherichia coli K-12 |
AAC76288.1 |
1789654 |
accD |
Escherichia coli K-12 |
AAC75376.1 |
1788655 |
|
|
|
|
accA |
Salmonella enterica |
CAD08690.1 |
16501513 |
accB |
Salmonella enterica |
CAD07894.1 |
16504441 |
accC |
Salmonella enterica, |
CAD07895.1 |
16504442 |
accD |
Salmonella enterica |
CAD07598.1 |
16503590 |
|
|
|
|
YMR207C |
Saccharomyces cerevisiae |
NP_013934.1 |
6323863 |
YNR016C |
Saccharomyces cerevisiae |
NP_014413.1 |
6324343 |
YGR037C |
Saccharomyces cerevisiae |
NP_011551.1 |
6321474 |
YKL182W |
Saccharomyces cerevisiae |
NP_012739.1 |
6322666 |
YPL231W |
Saccharomyces cerevisiae |
NP_015093.1 |
6325025 |
[0404] The product yields per C-mol of substrate of microbial cells synthesizing reduced
fermentation products such as 2,4-pentadienoate, 3-butene-1-ol, or 1,3-butadiene,
are limited by insufficient reducing equivalents in the carbohydrate feedstock. Reducing
equivalents, or electrons, can be extracted from synthesis gas components such as
CO and H
2 using carbon monoxide dehydrogenase (CODH) and hydrogenase enzymes, respectively.
The reducing equivalents are then passed to acceptors such as oxidized ferredoxins,
oxidized quinones, oxidized cytochromes, NAD(P)+, water, or hydrogen peroxide to form
reduced ferredoxin, reduced quinones, reduced cytochromes, NAD(P)H, H
2, or water, respectively. Reduced ferredoxin and NAD(P)H are particularly as they
can serve as redox carriers for various Wood-Ljungdahl pathway and reductive TCA cycle
enzymes.
[0405] Herein below the enzymes and the corresponding genes used for extracting redox from
synags components are described. CODH is a reversible enzyme that interconverts CO
and CO
2 at the expense or gain of electrons. The natural physiological role of the CODH in
ACS/CODH complexes is to convert CO
2 to CO for incorporation into acetyl-CoA by acetyl-CoA synthase. Nevertheless, such
CODH enzymes are suitable for the extraction of reducing equivalents from CO due to
the reversible nature of such enzymes. Expressing such CODH enzymes in the absence
of ACS allows them to operate in the direction opposite to their natural physiological
role (i.e., CO oxidation).
[0406] In
M.
thermoacetica, C.
hydrogenoformans, C. carboxidivorans P7, and several other organisms, additional CODH encoding genes are located outside of
the ACS/CODH operons. These enzymes provide a means for extracting electrons (or reducing
equivalents) from the conversion of carbon monoxide to carbon dioxide. The
M.
thermoacetica gene (Genbank Accession Number: YP_430813) is expressed by itself in an operon and
is believed to transfer electrons from CO to an external mediator like ferredoxin
in a "Ping-pong" reaction. The reduced mediator then couples to other reduced nicolinamide
adenine dinucleotide phosphate (NAD(P)H) carriers or ferredoxin-dependent cellular
processes (
Ragsdale, Annals of the New York Academy of Sciences 1125: 129-136 (2008)). The genes encoding the
C.
hydrogenoformans CODH-II and CooF, a neighboring protein, were cloned and sequenced (
Gonzalez and Robb, FEMS Microbiol Lett. 191:243-247 (2000)). The resulting complex was membrane-bound, although cytoplasmic fractions of CODH-II
were shown to catalyze the formation of NADPH suggesting an anabolic role (
Svetlitchnyi et al., J Bacteriol. 183:5134-5144 (2001)). The crystal structure of the CODH-II is also available (
Dobbek et al., Science 293:1281-1285 (2001)), Similar ACS-free CODH enzymes can be found in a diverse array of organisms including
Geobacter metallireducens GS- 15,
Chlorobium phaeobacteroides DSM 266,
Clostridium cellulolyticum H10,
Desulfovibrio desulfuricans subsp.
desulfuricans str. ATCC 27774,
Pelobacter carbinolicus DSM 2380, and
Campylobacter curvus 525.92.
Protein |
GenBank ID |
GI Number |
Organism |
CODH (putative) |
YP_430813 |
83590804 |
Moorella thermoacetica |
CODH-II (CooS-II) |
YP_3S8957 |
78044574 |
Carboxydothermus hydrogenoformans |
CooF |
YP_358938 |
78045112 |
Carboxydothermus hydrogenoformans |
CODH (putative) |
ZP_05390164.1 |
255523193 |
Clostridium carboxidivorans P7 |
CcarbDRAFT_0341 |
ZP_05390341.1 |
255523371 |
Clostridium caboxidivorans P7 |
CCarbDRAFT_1756 |
ZP_05391756.1 |
255524806 |
Clostridium caboxidivorans P7 |
CcarbDRAFT-2944 |
ZP_05392944.1 |
255526020 |
Clostridium caboxidivorans P7 |
CODH |
YP_384856.1 |
78223109 |
Geobacter metallireducens GS-15 |
Cpha266_0148 cytochrome c) |
YP_910642.1 |
119355998 |
Chlorobium phaeobacteroides DSM 266 |
Cpha26_0149 (CODH) |
YP_910643.1 |
119355999 |
Chlorobium phaeobacteroides DSM 266 |
Ccel_0438 |
YP_002S04800.1 |
220927891 |
Clostridium cellulolyticum H10 |
Ddes_0382 (CODH) |
YP_002478973.1 |
220903661 |
Desulfovibrio desulfuricans subsp. desulfuricans str. ATCC 27774 |
Dcles_0381 (CooC) |
YP_002478972.1 |
220903660 |
Desulfovibrio delsulfuricans subsp. desulfuricans str. ATCC 27774 |
Pcar_0057 (CODH) |
YP_355490.1 |
7791767 |
Pelobacter carbinolicus DSM 2380 |
Pcar_0058 (CooC) |
YP_355491.1 |
7791766 |
Pelobacter carbinolicus DSM 2380 |
Pcar_0058 (HypA) |
YP_355492.1 |
7791765 |
Pelobacter carbinolicus DSM 2380 |
CooS (CODH) |
YP_001407343.1 |
154175407 |
Campylobacter curvus 525.92 |
[0407] In some cases, hydrogenase encoding genes are located adjacent to a CODH. In
Rhodospirillum rubrum, the encoded CODH/hydrogenase proteins form a membrane-bound enzyme complex that has
been indicated to be a site where energy, in the form of a proton gradien, is generated
from the conversion of CO and H
2O to CO
2 and H
2 (
Fox et al., J Bacteriol. 178:6200-6208 (1996)). The CODH-I of
C. hydrogenoformans and its adjacent genes have been proposed to catalyze a similar functional role based
on their similarity to the
R. rubrum CODH/hydrogenase gene cluster (
Wu et al., PLoS Genet. 1:e65 (2005)). The
C.
hydrogenoformans CODH-I was also shown to exhibit intense CO oxidation and CO
2 reduction activities when linked to an electrode (
Parkin et. al., J Am.Chem.Soc. 129:10328-10329 (2007)). The protein sequences of exemplary CODH and hydrogenase genes can be identified
by the following GenBank accession numbers.
Protein |
GenBank ID |
GI Number |
Organism |
CODH-I (Coos-I) |
YP_360644 |
78043418 |
Carboxydothermus hydrogenoformans |
CooF |
YP_360645 |
78044791 |
Carboxydothermus hydrogenoformans |
HypA |
YP_360646 |
78044340 |
Carboxydothermus hydrogenoformans |
CooH |
YP_360647 |
78043871 |
Carboxydothermus hydrogenoformans |
CooU |
YP_360648 |
78044023 |
Carboxydothermus hydrogenoformans |
CooX |
YP_360649 |
78043124 |
Carboxydothermus hydrogenoformans |
CooL |
YP_360650 |
78043938 |
Carboxydothermus hydrogenoformans |
CooK |
YP_360651 |
78044700 |
Carboxydothermus hydrogenoformans |
CooM |
YP_360652 |
78043942 |
Carboxydothermus hydrogenoformans |
CooC |
YP_360654.1 |
78043296 |
Carboxydothermus hydrogenoformans |
CooA-1 |
YP-3 60655.1 |
78044021 |
Carboxydothermus hydrogenoformans |
CooL |
AAC45118 |
1515468 |
Rhodospirillum rubrum |
CooX |
AAC45119 |
1515469 |
Rhodospirillum rubrum |
CooU |
AAC45120 |
1515470 |
Rhodospirillum rubrum |
CooH |
AAC45121 |
1498746 |
Rhodospirillum rubrum |
CooF |
AAC45122 |
1498747 |
Rhodospirillum rubrum |
CODH(CooS) |
AAC45123 |
1498748 |
Rhodospirillum rubrum |
cooc |
AAC45124 |
1498749 |
Rhodospirillum rubrum |
CooT |
AAC45125 |
1498750 |
Rhodospirillum rubrum |
CooJ |
AAC45126 |
1498751 |
Rhodospirillum rubrum |
[0408] Native to
E. coli and other enteric bacteria are multiple genes encoding up to four hydrogenases (
Sawers, G., Antonie Van Leeuwenhocek 66:57-88 (1994);
Sawers et al., J Bacteriol. 164:1324-1331 (1985);
Sawers and Boxer, Eur.J Biochem. 156:265-275 (1986);
Sawers et al., J Bacteriol. 168:398-404 (1986)). Given the multiplicity of enzyme activities,
E. coli or another host organism can provide sufficient hydrogenase activity to split incoming
molecular hydrogen and reduce the corresponding acceptor.
E. coli possesses two uptake hydrogenases, Hyd-1 and Hyd-2, encoded by the
hyaABCDEF and
hypbOABCDEFG gene clusters, respectively (
Lukey et al., How E. coli is equipped to oxidize hydrogen under different redox conditions,
J Biol Chem published online Nov 16, 2009). Hyd-1 is oxygen-tolerant, irreversible, and is coupled to quinone reduction via
the hya
C cytochrome. Hyd-2 is sensitive to O
2, reversible, and transfers electrons to the periplasmic ferredoxin
hybA which, in turn, reduces a quinone via the
hybB integral membrane protein. Reduced quinones can serve as the source of electrons
for fumarate reductase in the reductive branch of the TCA cycle. Reduced ferredoxins
can be used by enzymes such as NAD(P)H:ferredoxin oxidoreductases to generate NADPH
or NADH. They can alternatively be used as the electron donor for reactions such as
pyruvate ferredoxin oxidoreductase, AKG ferredoxin oxidoreductase, and 5,1 0-methylene-H4folate
reductase.
Protein |
GenBank ID |
GI Number |
Organism |
HyaA |
AAC74057.1 |
1787206 |
Escherichia coli |
HyaB |
AAC74058.1 |
1787207 |
Escherichia coli |
HyaC |
AAC74059.1 |
1787208 |
Escherichia coli |
HyaD |
AAC74060.1 |
1787209 |
Escherichia coli |
HyaE |
AAC74061.1 |
1787210 |
Escherichia coli |
HyaF |
AAC74062.1 |
1787211 |
Escherichia coli |
HybO |
AAC76033.1 |
1789371 |
Escherichia coli |
HybA |
AAC76032.1 |
1789370 |
Escherichia coli |
HybB |
AAC.76031.1 |
2367183 |
Escherichia coli |
HybC |
AAC76030.1 |
1789368 |
Escherichia coli |
HybD |
AAC76029.1 |
1789367 |
Escherichia coli |
HybE |
AAC76028.1 |
1789366 |
Escherichia coli |
HybF |
AAC76027.1 |
1789365 |
Escherichia coli |
HybG |
AAC76026.1 |
1789364 |
Escherichia coli |
[0409] The hydrogen-lyase systems of
E. coli include hydrogenase 3, a membrane-bound enzyme complex using ferredoxin as an acceptor,
and hydrogenase 4 that also uses a ferredoxin acceptor. Hydrogenase 3 and 4 are encoded
by the
hyc and
hyf gene clusters, respectively. Hydrogenase has been shown to be a reversible enzyme
(
Maeda et al., Appl Microbiol Biotechnol 76(5): 103 5-42 (2007)). Hydrogenase activity in E.
coli is also dependent upon the expression of the
hyp genes whose corresponding proteins are involved in the assembly of the hydrogenase
complexes (
Jacobi et al., Arch.Microbiol 158:444-451 (1992);
Rangarajan et al., J. Bacteriol. 190:1447-1458 (2008)).
Protein |
GenBank ID |
GI Number |
Organism |
HycA |
Nip_417205 |
16130632 |
Escherichia coli |
HycB |
NP_417204 |
16130631 |
Escherichia coli |
HycC |
NP_417203 |
16130630 |
Escherichia coli |
HycD |
NP_417202 |
16130629 |
Escherichia coli |
HycE |
NP_417201 |
16130628 |
Escherichia coli |
HycF |
NP_417200 |
16130627 |
Escherichia coli |
HycG |
NP_417199 |
16130626 |
Escherichia coli |
HycH |
NP_417198 |
16130625 |
Escherichia coli |
HycI |
NP_417197 |
16130624 |
Escherichia coli |
Protein |
GenBank ID |
GI Number |
Organism |
HyfA |
NP_416976 |
90111444 |
Escherichia coli |
HyfB |
NP_416977 |
16130407 |
Escherichia coli |
HyfC |
NP_416978 |
90111445 |
Escherichia coli |
HyfD |
NP_416979 |
16130409 |
Escherichia coli |
HyfE |
NP_416980 |
16130410 |
Escherichia coli |
HyfF |
NP_416981 |
16130411 |
Escherichia coli |
HyfG |
NP_416982 |
16130412 |
Escherichia coli |
HyfH |
NP_416983 |
16130413 |
Escherichia coli |
HyfI |
NP_416984 |
16130414 |
Escherichia coli |
HyfJ |
NP_416985 |
90111446 |
Escherichia coli |
HyfR |
NP_416986 |
90111447 |
Escherichia coli |
Protein |
GenBank ID |
GI Number |
Organism |
HypA |
NP_417206 |
16130633 |
Escherichia coli |
HypB |
NP_417207 |
16130634 |
Escherichia coli |
HypC |
NP_417208 |
16130635 |
Escherichia coli |
HypD |
NP_417209 |
16130636 |
Escherichia coli |
HypE |
NP_417210 |
226524440 |
Escherichia coli |
HypF |
NP_417192 |
16130619 |
Escherichia coli |
[0411] Proteins in
M. thermoacetica whose genes are homologous to the
E. coli hyp genes are shown below.
Protein |
GenBankID |
GI Number |
Organism |
Moth_2175 |
YP_431007 |
83590998 |
Moorella thermoacetica |
Moth_2176 |
YP_431008 |
83590999 |
Moorella thermoacetica |
Moth_2177 |
YP_431009 |
83591000 |
Moorella thermoacetica |
Moth_2178 |
YP_431010 |
83591001 |
Moorella thermoacetica |
Moth_2179 |
YP_431011 |
83591002 |
Moorella thermoacetica |
Moth_2180 |
YP_431012 |
83591003 |
Moorella thermoacetica |
Moth_2181 |
YP_431013 |
83591004 |
Moorella thermoacetica |
[0412] Proteins in
M.
thermoacetica that are homologous to the
E. coli Hydrogenase 3 and/or 4 proteins are listed in the following table.
Protein |
GenBank ID |
GI Number |
Organism |
Moth_2182 |
YP_431014 |
83591005 |
Moorella thermoacetica |
Moth_2183 |
YP_431015 |
83591006 |
Moorella thermoacetica |
Moth_2184 |
YP_431016 |
83591007 |
Moorella thermoacetica |
Moth_2185 |
YP_431017 |
83591008 |
Moorella thermoacetica |
Moth_2186 |
YP_431018 |
83591009 |
Moorella thermoacetica |
Moth_2187 |
YP_431019 |
83591010 |
Moorella thermoacetica |
Moth_2188 |
YP_431020 |
83591011 |
Moorella thermoacetica |
Moth_2189 |
YP_411021 |
83591012 |
Moorella thermoacetica |
Moth_2190 |
YP_431022 |
83591013 |
Moorella thermoacetica |
Moth_2191 |
YP_431023 |
83591014 |
Moorella thermoacetica |
Moth_2192 |
YP_431024 |
83591015 |
Moorella thermoacetica |
[0413] In addition, several gene cluster encoding hydrogenase functionality are present
in
M. thermoacetica and their corresponding protein sequences are provided below.
Protein |
GenBank ID |
GI Number |
Organism |
Moth_0439 |
YP_429313 |
83589304 |
Moorella thermoacetica |
Moth_440 |
YP_429314 |
83589305 |
Moorella thermoacetica |
Moth_0441 |
YP_429315 |
83589306 |
Moorella thermoacetica |
Moth_0442 |
YP_429316 |
83589307 |
Moorella thermoacetica |
Moth_0809 |
YP_429670 |
83589661 |
Moorella thermoacetica |
Moth_0810 |
YP_429671 |
83589662 |
Moorella thermoacetica |
Moth_0811 |
YP_429672 |
83589663 |
Moorella thermoacetica |
Moth_0812 |
YP_429673 |
83589664 |
Moorella thermoacetica |
Moth_0814 |
YP_429674 |
83589665 |
Moorella thermoacetica |
Moth_0815 |
YP_429675 |
83589666 |
Moorella thermoacetica |
Moth_0816 |
YP_429676 |
83589667 |
Moorella thermoacetica |
Moth_1193 |
YP_430050 |
83590041 |
Moorella thermoacetica |
Moth_1194 |
YP_430051 |
83590042 |
Moorella thermoacetica |
Moth_1195 |
YP_430052 |
83590043 |
Moorella thermoacetica |
Moth_1196 |
YP_430053 |
83590044 |
Moorella thermoacetica |
Moth_1717 |
YP_430562 |
83590553 |
Moorella thermoacetica |
Moth_1718 |
YP_430563 |
83590554 |
Moorella thermoacetica |
Moth_1719 |
YP_430S64 |
83590555 |
Moorella thermoacetica |
Moth_1883 |
YP_430726 |
83590717 |
Moorella thermoacetica |
Moth_1884 |
YP_430727 |
83590718 |
Moorella thermoacetica |
Moth_1885 |
YP_430728 |
83590719 |
Moorella thermoacetica |
Moth_1886 |
YP_430729 |
83590720 |
Moorella thermoacetica |
Moth_1887 |
YP_430730 |
83590721 |
Moorella thermoacetica |
Moth_1888 |
YP_430731 |
83590722 |
Moorella thermoacetica |
Moth_1452 |
YP_430305 |
83590296 |
Moorella thermoacetica |
Moth_1453 |
YP_430306 |
83590297 |
Moorella thermoacetica |
Moth_1454 |
YP_430307 |
83590298 |
Moorella thermoacetica |
[0414] Ralstonia eutropha H16 uses hydrogen as an energy source with oxygen as a terminal electron acceptor.
Its membrane-bound uptake [NiFe]-hydrogenase is an "O
2-tolerant" hydrogenase (
Cracknell, et al. Proc Nat Acad Sci, 106(49) 20681-20686 (2009)) that is periplasmically-oriented and connected to the respiratory chain via a b-type
cytochrome (
Schink and Schiegel, Biochim. Biophys. Acta, 567, 315-324 (1979);
Bernhard et al., Eur. J. Biochem. 248, 179-186 (1997)).
R.
eutropha also contains an O
2-tolerant soluble hydrogenase encoded by the
Hox operon which is cytoplasmic and directly reduces NAD+ at the expense of hydrogen.
(
Schneider and Schlegel, Biochim. Biophys. Acta 452, 66-80 (1976);
Burgdorf, J. Bact. 187(9) 3122-3132(2005)). Soluble hydrogenase enzymes are additionally present in several other organisms
including
Geobacter sulfurreducens (
Coppi, Microbiology 151, 1239-1254 (2005)),
Synechocysistis str. PCC 6803 (
Germer, J. Biol. Chem., 284(52), 36462-30472 (2009)), and
Thiocapsa roseopersicina (
Rakhely, Appl. Environ. Microbiol. 70(2) 722-728 (2004)), The
Synechocystis enzyme is capable of generating NADPH from hydrogen. Overexpression of both the
Hox operon from
Synechocystis str. PCC 6803 and the accessory genes encoded by the
Hyp operon from
Nostoc sp. PCC 7120 led to increased hydrogenase activity compared to expression of the
Hox genes alone (
Germer, J. Biol. Chem. 284(52), 36462-36472 (2009)).
Protein |
GenBank ID |
GI Number |
Organism |
HoxF |
NP_942727.1 |
38637753 |
Ralstonia eutropha H16 |
HoxU |
NP_942728.1 |
38637754 |
Ralstonia eutropha H16 |
HoxY |
NP_942729.1 |
38637755 |
Ralstonia eutropha H16 |
HoxH |
NP_942730.1 |
38637756 |
Ralstonia eutropha H16 |
HoxW |
NP_942731.1 |
38637757 |
Ralstonia eutropha H16 |
HoxI |
NP_942732.1 |
38637758 |
Ralstonia eutropha H16 |
HoxE |
NP_953767.1 |
39997816 |
Geobacter sulfurreducens |
HoxF |
NP_953766.1 |
39997815 |
Geobacter sulfurreducens |
HoxU |
NP_953765.1 |
39997814 |
Geobacter sulfurreducens |
HoxY |
NP_953764.1 |
39997813 |
Geobacter sulfurreducens |
HoxH |
NP_953763.1 |
39997812 |
Geobacter sulfurreducens |
GSU2717 |
NP_953762.1 |
39997811 |
Geobacter sulfurreducens |
HoxE |
NP_441418.1 |
16330690 |
Synechocystis str. PCC 6803 |
HoxF |
NP_441417.1 |
16330689 |
Synechocystis str. PCC 6803 |
Unknown function |
NP_441416.1 |
16330688 |
Synechocystis str. PCC 6803 |
HoxU |
NP_441415.1 |
16330687 |
Synechocystis str. PCC 6803 |
HoxY |
NP_441414.1 |
16330686 |
Synechocystis str. PCC 6803 |
Unknown function |
NP_441413.1 |
16330685 |
Synechocystis str. PCC 6803 |
Unknown function |
NP_441412.1 |
16330684 |
Synechocystis str. PCC 6803 |
HoxH |
NP_441411.1 |
16330683 |
Synechocystis str. PCC 6803 |
HypF |
NP_484737.1 |
17228189 |
Nostoc sp. PCC 7120 |
HypC |
NP_484738.1 |
17228190 |
Nostoc sp. PCC 7120 |
HypD |
NP_484739.1 |
17228191 |
Nostoc sp. PCC 7120 |
Unknown function |
NP_484740.1 |
17228192 |
Nostoc sp. PCC 7120 |
HypE |
NP_484741.1 |
17228193 |
Nostoc sp. PCC 7120 |
HypA |
NP_484742.1 |
17228194 |
Nostoc sp. PCC 7120 |
HypB |
NP_484743.1 |
17228195 |
Nostoc sp. PCC 7120 |
Hox1E |
AAP50519.1 |
37787351 |
Thiocapsa roseopersicina |
Hox1F |
AAP50520.1 |
37787352 |
Thiocapsa roseopersicina |
Hox1U |
AAP50521.1 |
37787353 |
Thiocapsa roseopersicina |
Hox1Y |
AAP50522.1 |
37787354 |
Thiocapsa roseopersicina |
Hox1H |
AAP50523.1 |
37787355 |
Thiocapsa roseopersicina |
[0415] Several enzymes and the corresponding genes used for fixing carbon dioxide to either
pyruvate or phosphoenolpyruvate to form the TCA cycle intermediates, oxaloacetate
or malate are described below.
[0417] An alternative enzyme for converting phosphoenolpyruvate to oxaloacetate is PEP carboxykinase,
which simultaneously forms an ATP while carboxylating PEP. In most organisms PEP carboxykinase
serves a gluconeogenic function and converts oxaloacetate to PEP at the expense of
one ATP.
S. cerevisiae is one such organism whose native PEP carboxykinase,
PCK1, serves a gluconeogenic role (
Valdes-Hevia et al., FEBS Lett. 258:313-316 (1989).
E. coli is another such organism, as the role of PEP carboxykinase in producing oxaloacetate
is believed to be minor when compared to PEP carboxylase, which does not form ATP,
possibly due to the higher K
m for bicarbonate of PEP carboxykinase (
Kim et al., Appl. Environ. Microbiol. 70:1238-1241 (2004)). Nevertheless, activity of the native
E. coli PEP carboxykinase from PEP towards oxaloacetate has been recently demonstrated in
ppc mutants of
E. coli K-12 (
Kwon et al., J. Microbiol. Biotechnol. 16:1448-1452 (2006)). These strains exhibited no growth defects and had increased succinate production
at high NaHCO
3 concentrations. Mutant strains of
E. coli can adopt Pck as the dominant CO2-fixing enzyme following adaptive evolution (Zhang
et al. 2009). In some organisms, particularly rumen bacteria, PEP carboxykinase is
quite efficient in producing oxaloacetate from PEP and generating ATP. Examples of
PEP carboxykinase genes that have been cloned into
E. coli include those from
Mannheimia succiniciproducens (
Lee et al., Biotechnol. Bioprocess Eng. 7:95-99 (2002)),
Anaerobiospirillum succiniciproducens (
Laivenieks et al., Appl. Environ. Microbiol. 63:2273-2280 (1997), and
Actinobacillus succinogenes (Kim et al.
supra). The PEP carboxykinase enzyme encoded by
Haemophilus influenza is effective at forming oxaloacetate from PEP.
Protein |
GenBank ID |
GI Number |
Organism |
PCK1 |
NP_013023 |
6322950 |
Saccharomyces cerevisiae |
pck |
NP_417862.1 |
16131280 |
Escherichia coli |
pckA |
YP_089485.1 |
52426348 |
Mannheimia succiniciproducens |
pckA |
009460.1 |
3122621 |
Anaerobiospirillum succiniciproducens |
pckA |
Q6W6X5 |
75440571 |
Actinobacillus succinogenes |
pcKA |
P43923.1 |
1172573 |
Haemophilus influenza |
[0419] Malic enzyme can be applied to convert CO
2 and pyruvate to malate at the expense of one reducing equivalent. Malic enzymes for
this purpose can include, without limitation, malic enzyme (NAD-dependent) and malic
enzyme (NADP-dependent). For example, one of the
E. coli malic enzymes (
Takeo, J. Biochem. 66:379-387 (1969)) or a similar enzyme with higher activity can be expressed to enable the conversion
of pyruvate and CO
2 to malate. By fixing carbon to pyruvate as opposed to PEP, malic enzyme allows the
high-energy phosphate bond from PEP to be conserved by pyruvate kinase whereby ATP
is generated in the formation of pyruvate or by the phosphotransferase system for
glucose transport. Although malic enzyme is typically assumed to operate in the direction
of pyruvate formation from malate, overexpression of the NAD-dependent enzyme, encoded
by maeA, has been demonstrated to increase succinate production in
E. coli while restoring the lethal Δpfl-AldhA phenotype under anaerobic conditions by operating
in the carbon-fixing direction (
Stols and Donnelly, Appl. Environ. Microbiol. 63(7) 2695-2701 (1997)). A similar observation was made upon overexpressing the malic enzyme from
Ascaris suum in
E. coli (
Stols et al., Appl. Biochem. Biotechnol. 63-65(1), 153-158 (1997)). The second
E.
coli malic enzyme, encoded by
maeB, is NADP-dependent and also decarboxylates oxaloacetate and other alpha-keto acids
(
Iwakura et al., J. Biochem. 85(5):1355-65 (1979)).
Protein |
GenBank ID |
GI Number |
Organism |
maeA |
NP_415996 |
90111281 |
Escherichia coli |
maeB |
NP_416958 |
16130388 |
Escherichia coli |
NAD-ME |
P27443 |
126732 |
Ascaris suum |
[0420] The enzymes used for converting oxaloacetate (formed from, for example, PEP carboxylase,
PEP carboxykinase, or pyruvate carboxylase) or malate (formed from, for example, malic
enzyme or malate dehydrogenase) to succinyl-CoA via the reductive branch of the TCA
cycle are malate dehydrogenase, fumarate dehydratase (fumarase), fumarate reductase,
and succinyl-CoA transferase. The genes for each of the enzymes are described herein
above.
[0421] Enzymes, genes and methods for engineering pathways from succinyl-CoA to various
products into a microorganism are now known in the art. The additional reducing equivalents
obtained from CO and/or H
2, as disclosed herein, improve the yields of
2,4-pentadienoate, 3-butene-1-ol, or 1,3-butadiene when utilizing carbohydrate-based feedstock. For example,
2,4-pentadienoate, 3-butene-1-ol, or 1,3-butadiene can be produced from succinyl-CoA via
pathways exemplified in Figure 20. Exemplary enzymes for the conversion succinyl-CoA to
2,4-pentadienoate, 3-butene-1-ol, or 1,3-butadiene include succinyl-CoA:acetyl-CoA acyltransferase, 3-oxoadipyl-CoA transferase, synthetase
or hydrolase, 3-oxoadipate dehydrogenase, 2-fumarylacetate decarboxylase, 3-oxopent-4-enoate
reductase, 3-hydroxypent-4-enoate dehydratase, 3-oxoadipyl-CoA reductase, 3-hydroxyadipyl-CoA
transferase, synthetase or hydrolase, 3-hydroxyadipate dehydrogenase, 3-hydroxyhex-4-enedioate
decarboxylase, 3-oxoadipate reductase, 2-fumarylacetate reductase, 3-hydroxypent-4-enoate
decarboxylase, 2,4-pentadienoate decarboxylase.
[0422] Enzymes, genes and methods for engineering pathways from glycolysis intermediates
to various products into a microorganism are known in the art. The additional reducing
equivalents obtained from CO and H
2, as described herein, improve the yields of all these products on carbohydrates.
EXAMPLE X
Methods for Handling CO and Anaerobic Cultures
[0423] This example describes methods used in handling CO and anaerobic cultures.
[0424] A. Handling of CO in small quantities for assays and small cultures. CO is an odorless, colorless and tasteless gas that is a poison. Therefore, cultures
and assays that utilized CO required special handling. Several assays, including CO
oxidation, acetyl-CoA synthesis, CO concentration using myoglobin, and CO tolerance/utilization
in small batch cultures, called for small quantities of the CO gas that were dispensed
and handled within a fume hood. Biochemical assays called for saturating very small
quantities (<2 mL) of the biochemical assay medium or buffer with CO and then performing
the assay. All of the CO handling steps were performed in a fume hood with the sash
set at the proper height and blower turned on; CO was dispensed from a compressed
gas cylinder and the regulator connected to a Schlenk line. The latter ensures that
equal concentrations of CO were dispensed to each of several possible cuvettes or
vials. The Schlenk line was set up containing an oxygen scrubber on the input side
and an oil pressure release bubbler and vent on the other side. Assay cuvettes were
both anaerobic and CO-containing. Threfore, the assay cuvettes were tightly sealed
with a rubber stopper and reagents were added or removed using gas-tight needles and
syringes. Secondly, small (∼50 mL) cultures were grown with saturating CO in tightly
stoppered serum bottles. As with the biochemical assays, the CO-saturated microbial
cultures were equilibrated in the fume hood using the Schlenk line setup. Both the
biochemical assays and microbial cultures were in portable, sealed containers and
in small volumes making for safe handling outside of the fume hood. The compressed
CO tank was adjacent to the fume hood.
[0425] Typically, a Schlenk line was used to dispense CO to cuvettes, each vented. Rubber
stoppers on the cuvettes were pierced with 19 or 20 gage disposable syringe needles
and were vented with the same. An oil bubbler was used with a CO tank and oxygen scrubber.
The glass or quartz spectrophotometer cuvettes have a circular hole on top into which
a Kontes stopper sleeve, Sz7 774250-0007 was fitted. The CO detector unit was positioned
proximal to the fume hood.
[0426] B. Handling of CO in larger quantities fed to large-scale cultures. Fermentation cultures are fed either CO or a mixture of CO and H
2 to simulate syngas as a feedstock in fermentative production. Therefore, quantities
of cells ranging from 1 liter to several liters can include the addition of CO gas
to increase the dissolved concentration of CO in the medium. In these circumstances,
fairly large and continuously administered quantities of CO gas are added to the cultures.
At different points, the cultures are harvested or samplers removed. Alternatively,
cells are harvested with an integrated continuous flow centrifuge that is part of
the fermenter.
[0427] The fermentative processes are carried out under anaerobic conditions. In some cases,
it is uneconomical to pump oxygen or air into fermenters to ensure adequate oxygen
saturation, to provide a respiratory environment. In addition, the reducing power
generated during anaerobic fermentation may be in product formation rather than respiration.
Furthermore, many of the enzymes for various pathways are oxygen-sensitive to varying
degrees. Classic acetogens such as
M.
Thermoacetica are obligate anaerobes and the enzymes in the Wood-Ljungdahl pathway are highly sensitive
to irreversible inactivation by molecular oxygen. While there are oxygen-tolerant
acetogens, the repertoire of enzymes in the Wood-Ljungdahl pathway might be incompatible
in the presence of oxygen because most are metallo-enzymes, key components are ferredoxins,
and regulation can divert metabolism away from the Wood-Ljungdahl pathway to maximize
energy acquisition. At the same time, cells in culture act as oxygen scavengers that
moderate the need for extreme measures in the presence of large cell growth.
[0428] C. Anaerobic cliamber and conditions. Exemplary anaerobic chambers are available commercially (see, for example, Vacuum
Atmospheres Company, Hawthorne CA; MBraun, Newburyport MA). Conditions included an
O
2 concentration of 1 ppm or less and 1 atm pure N
2. In one example, 3 oxygen scrubbers/catalyst regenerators were used, and the chamber
included an O
2 electrode (such as Teledyne; City of Industry CA). Nearly all items and reagents
were cycled four times in the airlock of the chamber prior to opening the inner chamber
door. Reagents with a volume >5mL were sparged with pure N
2 prior to introduction into the chamber. Gloves are changed twice/yr and the catalyst
containers were regenerated periodically when the chamber displays increasingly sluggish
response to changes in oxygen levels. The chamber's pressure was controlled through
one-way valves activated by solenoids. This feature allowed setting the chamber pressure
at a level higher than the surroundings to allow transfer of very small tubes through
the purge valve.
[0429] The anaerobic chambers achieved levels Of O
2 that were consistently very low and were needed for highly oxygen sensitive anaerobic
conditions. However, growth and handling of cells not usually require such precautions.
In an alternative anaerobic chamber configuration, platinum or palladium can be used
as a catalyst that requires some hydrogen gas in the mix. Instead of using solenoid
valves, pressure release can be controller by a bubbler. Instead of using instrument-based
O
2 monitoring, test strips can be used instead.
[0430] D. Anaerobic microbiology. Small cultures were handled as described above for CO handling. In particular, serum
or media bottles are fitted with thick rubber stoppers and aluminum crimps are employed
to seal the bottle. Medium, such as Terrific Broth, is made in a conventional manner
and dispensed to an appropriately sized serum bottte. The bottles are sparged with
nitrogen for ~30 min of moderate bubbling. This removes most of the oxygen from the
medium and, after this step, each bottle is capped with a rubber stopper (such as
Bellco 20 mm septum stoppers; Bellco, Vineland, NJ) and crimp-sealed (Bellco 20 mm).
Then the bottles of medium are autoclaved using a slow (liquid) exhaust cycle. At
least sometimes a needle can be poked through the stopper to provide exhaust during
autoclaving; the needle needs to be removed immediately upon removal from the autoclave.
The sterile medium has the remaining medium components, for example buffer or antibiotics,
added via syringe and needle. Prior to addition of reducing agents, the bottles are
equilibrated for 30 - 60 minutes with nitrogen (or CO depending upon use). A reducing
agent such as a 100 x 150 mM sodium sulfide, 200 mM cysteine-HCl is added. This is
made by weighing the sodium sulfide into a dry beaker and the cysteine into a serum
bottle, bringing both into the anaerobic chamber, dissolving the sodium sulfide into
anaerobic water, then adding this to the cysteine in the serum bottle. The bottle
is stoppered immediately as the sodium sulfide solution generates hydrogen sulfide
gas upon contact with the cysteine. When injecting into the culture, a syringe filter
is used to sterilize the solution. Other components are added through syringe needles,
such as B12 (10 µM cyanocobalamin), nickel chloride (NiCl
2, 20 microM final concentration from a 40 mM stock made in anaerobic water in the
chamber and sterilized by autoclaving or by using a syringe filter upon injection
the culture), and ferrous ammonium sulfate (final concentration needed is 100 µM-made
as 100-1000x stock solution in anaerobic water in the chamber and sterilized by autoclaving
or by using a syringe filter upon injection into the culture). To facilitate faster
growth under anaerobic conditions, the 1 liter bottles were inoculated with 50 mL
of a preculture grown anaerobically. Induction of the pA1-lacO1 promoter in the vectors
was performed by addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final
concentration of 0.2 mM and was carried out for about 3 hrs.
[0431] Large cultures can be grown in larger bottles using continuous gas addition while
bubbling. A rubber stopper with a metal bubbler is placed in the bottle after medium
addition and sparged with nitrogen for 30 minutes or more prior to setting up the
rest of the bottle. Each bottle is put together such that a sterile filter will sterilize
the gas bubbled in and the hoses on the bottles are compressible with small C clamps
. Medium and cells are stirred with magnetic stir bars. Once all medium components
and cells are added, the bottles are incubated in an incubator in room air but with
continuous nitrogen sparging into the bottles.
EXAMPLE XI
CO oxidation (CODH) Assay
[0432] This example describes assay methods for measuring CO oxidation (CO dehydrogenase;
CODH).
[0433] The 7 gene CODH/ACS operon of
Moorella thermoacetica was cloned into
E. coli expression vectors. The intact ~10 kbp DNA fragment was cloned, and it is likely
that some of the genes in this region are expressed from their own endogenous promoters
and all contain endogenous ribosomal binding sites. These clones were assayed for
CO oxidation, using an assay that quantitatively measures CODH activity. Antisera
to the
M. thermoacetica gene products was used for Western blots to estimate specific activity.
M.
thermoacetica is Gram positive, and ribosome binding site elements are expected to work well in
E. coli. This activity, described below in more detail, was estimated to be ~1/50th of the
M.
thermoacetica specific activity. It is possible that CODH activity of recombinant
E. coli cells could be limited by the fact that
M.
thermoacetica enzymes have temperature optima around 55°C. Therefore, a mesophilic CODH/ACS pathway
could be advantageous such as the close relative of
Moorella that is mesophilic and does have an apparently intact CODH/ACS operon and a Wood-Ljungdahl
pathway,
Desulfitobacterium hafniense. Acetogens as potential host organisms include, but are not limited to,
Rhodospirillum rubrum, Moorella thermoacetica and Desulfitobacterium hafniense.
[0434] CO oxidation is both the most sensitive and most robust of the CODH/ACS assays. It
is likely that an
E.
coli-based syngas using system will ultimately need to be about as anaerobic as
Clostridial (i.e.,
Moorella) systems, especially for maximal activity. Improvement in CODH should be possible
but will ultimately be limited by the solubility of CO gas in water.
[0435] Initially, each of the genes was cloned individually into expression vectors. Combined
expression units for multiple subunits/1 complex were generated. Expression, in
E.
coli at the protein level was determined. Both combined
M. thermoacetica CODH/ACS operons and individual expression clones were made.
[0436] CO oxidation assay. This assay is one of the simper, reliable, and more versatile
assays of enzymatic activities within the Wood-Ljungdahl pathway and tests CODH (
Seravalli et al., Biochemistry 43:3944-3955 (2004)). A typical activity of
M. thermoacetica CODH specific activity is 500 U at 55°C or ~60U at 25°C. This assay employs reduction
of methyl viologen in the presence of CO. This is measured at 578 nm in stoppered,
anaerobic, glass cuvettes.
[0437] In more detail, glass rubber stoppered cuvettes were prepared after first washing
the cuvette four times in deionized water and one time with acetone. A small amount
of vacuum grease was smeared on the top of the rubber gasket. The cuvette was gassed
with CO, dried 10 min with a 22 Ga. needle plus an exhaust needle. A volume of 0.98
mlL of reaction buffer (50 mM Hepes, pH 8.5,2mM dithiothreitol (DTT) was added using
a 22 Ga. needle, with exhaust needled, and 100%CO. Methyl viologen. (CH
3 viologen) stock was 1 M in water. Each assay used 20 microliters for 20 mM final
concentration. When methyl viologen was added, an 18 Ga needle (partial) was used
as a jacket to facilitate use of a Hamilton syringe to withdraw the CH
3 viologen. 4 -5 aliquots were drawn up and discarded to wash and gas equilibrate the
syringe. A small amount of sodium dithionite (0.1 M stock) was added when making up
the CH
3 viologen stock to slightly reduce the CH
3 viologen. The temperature was equilibrated to 55°C in a heated Olis spectrophotometer
(Bogart GA). A blank reaction (CH
3 vioiogen + buffer) was run first to measure the base rate of CH
3 viologen reduction. Crude
E.
coli cell extracts of ACS90 and ACS91 (CODH-ACS operon of
M. thermoacetica with and without, respectively, the first
cooC). 10 microliters of extract were added at a time, mixed and assayed. Reduced CH
3 viologen turns purple. The results of an assay are shown in Table I.
Table I. Crude extract CO Oxidation Activities.
ACS90 |
7.7 mg/ml |
ACS91 |
11.8 mg/ml |
Mta98 |
9.8 mg/ml |
Mta99 |
11.2 mg/ml |
|
|
|
|
|
Extract |
Vol |
OD/ |
U/ml |
U/mg |
ACS90 |
10 microliters |
0.073 |
0.376 |
0.049 |
ACS91 |
10 microliters |
0.096 |
0.494 |
0.042 |
Mta99 |
10 microliters |
0.0031 |
0.016 |
0.0014 |
ACS90 |
10 microliters |
0.099 |
00.51 |
0.066 |
Mta99 |
25 microliters |
0.012 |
0.025 |
0.0022 |
ACS91 |
25 microliters |
0.215 |
0.443 |
0.037 |
Mta98 |
25 microliters |
0.019 |
0.039 |
0.004 |
ACS91 |
10 microliters |
0.129 |
0.66 |
0.056 |
|
|
|
|
|
Averages |
|
|
|
|
ACS90 |
0.057 U/mg |
|
|
|
ACS91 |
0.045 U/mg |
|
|
|
Mta99 |
0.0018 U/mg |
|
|
|
[0438] Mta98/Mta99 are
E. coli MG1655 strains that express methanol methyltransferase genes from
M. thermoacetia and, therefore, are negative controls for the ACS90 ACS91
E.
coli strains that contain
M. thermoacetica CODH operons.
[0439] If ~ 1% of the cellular protein is CODH, then these figures would be approximately
100X less than the 500 U/mg activity of pure
M. thermoacetica CODH. Actual estimated based on Western blots are 0.5% of the cellular protein, so
the activity is about 50X less than for
M.
thermoacetica CODH. Nevertheless, this experiment demonstrates CO oxidation activity in recombinant
E. coli with a much smaller amount in the negative controls. The small amount of CO oxidation
(CH
3 viologen reduction) seen in the negative controls indicates that
E. coli may have a limited ability to reduce CH
3 viologen.
[0440] To estimate the final concentrations of CODH and Mtr proteins, SDS-PAGE followed
by Western blot analyses were performed on the same cell extracts used in the CO oxidation,
ACS, methyltransferase, and corrinoid Fe-S assays. The antisera were polyclonal to
purified
M.
thermoacetica CODH-ACS and Mtr proteins and were visualized using an alkaline phosphatase-linked
goat-anti-rabbit secondary antibody. The Westerns were performed and results are shown
in Figure 24. The amounts of CODH in ACS90 and ACS91 were estimated at 50 ng by comparison
to the control lanes. Expression of CODH-ACS operon genes including 2 CODH subunits
and the methyltransferase were confirmed via Western blot analysis. Therefore, the
recombinant
E.
coli cells express multiple components of a 7 gene operon. In addition, both the methyltransferase
and corrinoid iron sulfur protein were active in the same recombinant
E. coli cells. These proteins are part of the same operon cloned into the same cells.
[0441] The CO oxidation assays were repeated using extracts of
Moorella thermoacetica cells for the positive controls. Though CODH activity in
E.
coli ACS90 and ACS91 was measurable, it was at about 130 - 150 X lower than the
M.
thermoacetica control. The results of the assay are shown in Figure 25. Briefly, cells (
M.
thermoacetica or
E. coli with the CODH/ACS operon; ACS90 or ACS91 or empty vector: pZA33S) were grown and
extracts prepared as described above. Assays were performed as described above at
55°C at various times on the day the extracts were prepared. Reduction of methylviologen
was followed at 578 nm over a 120 sec time course.
[0442] These results describe the CO oxidation (CODH) assay and results. Recombinant
E.
coli cells expressed CO oxidation activity as measured by the methyl viologen reduction
assay.
EXAMPLE XII
E. coli CO Tolerance Experiment and CO Concentration Assay. (myoglobin assay)
This example describes the tolerance of E. coli for high concentrations of CO.
[0443] To test whether or not
E. coli can grow anaerobically in the presence of saturating amounts of CO, cultures were
set up in 120 ml serum bottles with 50 ml of Terrific Broth medium (plus reducing
solution, NiCl
2);, Fe(II)NH
4SO
4, cyanocobalamin, IPTG, and chloramphenicol) as described above for anaerobic microbiology
in small volumes. One half of these bottles were equilibrated with nitrogen gas for
30 min. and one half was equilibrated with CO gas for 30 min. An empty vector (pZA33)
was used as a control, and cultures containing the pZA33 empty vector as well as both
ACS90 and ACS91 were tested with both N
2 and CO. All were inoculated and grown for 36 hrs with shaking (250 rpm) at 37°C.
At the end of the 36 hour period, examination of the flasks showed high amounts of
growth in all. The bulk of the observed growth occurred overnight with a long lag.
[0444] Given that all cultures appeared to grow well in the presence of CO, the final CO
concentrations were confirmed. This was performed using an assay of the spectral shift
of myoglobin upon exposure to CO. Myoglobin reduced with sodium dithionite has an
absorbance peak at 435 nm; this peak is shifted to 423 nm with CO. Due to the low
wavelength and need to record a whole spectrum from 300 nm on upwards, quartz cuvettes
must be used. CO concentration is measured against a standard curve and depends upon
the Henry's Law constant for CO of maximum water solubility = 970 micromolar at 20°C
and atm.
[0445] For the myoglobin test of CO concentration, cuvettes were washed 10X with water,
1X with acetone, and then stoppered as with the CODH assay. N
2 was blown into the cuvettes for ∼10 min. A volume of 1 ml of anaerobic buffer (HEPES,
pH 8.0, 2mM DTT) was added to the blank (not equilibrated with CO) with a Hamilton
syringe. A volume of 10 microliter myoglobin (∼1 mM-can be varied, just need a fairly
large amount) and 1 microliter dithionite (20 mM stock) were added. A CO standard
curve was made using CO saturated buffer added art 1 microliter increments. Peak height
and shift was recorded for each increment. The cultures tested were pZA33/CO. ACS90/CO,
and ACS91/CO. Each of these was added in 1 microliter increments to the same cuvette.
Midway through the experiment a second cuvette was set up and used. The results are
shown in Table II.
[0446] Table II. Carbon Monoxide Concentrations, 36 hrs.
Strain and Growth Conditions pZA33-CO |
Final CO concentration(micromolar) 930 |
|
|
ACS90-CO |
638 |
|
494 |
|
734 |
|
883 |
ave |
687 |
SD |
164 |
|
|
ACS91-CO |
728 |
812 |
812 |
|
760 |
|
611 |
ave. |
728 |
SD |
85 |
[0447] The results shown in Table II indicate that the cultures grew whether or not a strain
was cultured in the presence of CO or not. These results indicate that
E. coli can tolerate exposure to CO under anaerobic conditions and that
E. coli cells expressing the CODH-ACS operon can metabolize some of the CO.
[0448] These results demonstrate that
E. coli cells, whether expressing CODH/ACS or not, were able to grow in the presence of saturating
amounts of CO. Furthermore, these grew equally well as the controls in nitrogen in
place of CO. This experiment demonstrated that laboratory strains of
E.
coli are insensitive to CO at the levels achievable in a syngas project performed at normal
atmospheric pressure. In addition, preliminary experiments indicated that the recombinant
E. coli cells expressing CODH/ACS actually consumed some CO, probably by oxidation to carbon
dioxide.
Example XIII
3-Hydroxyacid decarboxylase enzymes for formation of 1,3-butadiene and 3-butene-1-ol
[0449] 3-Hydroxyacid decarboxylase enzymes catalyze the ATP-driven decarboxylation of 3-hydroxyacids
to alkene derivatives. 3-Hyroxyacid decarboxylase enzymes have recently been described
that catalyze the formation of isobutylene, propylene and ethylene (
WO 2010/001078 and
Gogerty and Bobik, Appl. Environ. Microbiol., p. 8004-8010, Vol. 76. No. 24 (2010)). We propose here a the application of similar enzymes to catalyze the conversion
of 3-hydroxypent-4-enoate (3HP4) to 1,3-butadiene, shown in Figure 16 and 3,5-dihydroxypentanoate
to 3-butene-1-ol , shown in Figures 17. The 3-butene-1-ol product can then be converted
to butadiene via chemical dehydration or biological dehydration via a 3-butene-1-ol
dehydratase, enzyme.
[0450] Conversion of 3-hydroxypent-4-enoate to butadiene is carried out by a 3-hydroxypent-4-enoate
decarboxylase. Similarly, conversion of 3,5-dihydroxypentanoate to 3-butene-1-ol is
carried out by a 3,5-dihydroxypentanoate decarboxylase. Such enzymes many share similarity
to mevalonate pyrophosphate decarboxylase or diphosphomevalonate decarboxylase enzymes.
One potential 3-hydroxypent-4-enoate decarboxylase is the
Saccharomyces cerevisae mevalonate diphosphate decarboxylase (
ScMDD or ERG 19) which was shown to convert 3-hydroxy-3-methylbutyrate (3-HMB) to isobutene
(
Gogerty and Bobik, 2010, Appl. Environ. Microbiol., p. 8004-8010, Vol. 76, No. 24). Two improved variants of the enzyme,
ScMDD1 (I145F) and
ScMDD2 (R74H), were demonstrated to achieve 19-fold and 38-fold increases compared to
the wild-type His-tagged enzyme. ERG19 and additional enzymes candidates are provided
below.
Enzyme |
GI |
Accession Number |
Organism |
ERG19 |
6324371 |
NP_014441.1 |
Saccharomyces cerevisiae S288C |
CAWG_01359 |
238879484 |
EEQ43122.1 |
Candida albicans WO-1 |
ANI_1_332184 |
145256805 |
XP_001401521.1 |
Aspergillus niger CBS 513.88 |
MVD |
4505289 |
NP_002452.1 |
Homo sapiens |
Ahos_1490 |
332797171 |
YP_004458671.1 |
Acidianus hospitalis W1 |
SSO2989 |
15899699 |
NP_344304.1 |
Sulfolobus solfataricus P2 |
UNLARM2_0386 |
255513677 |
EET89942.1 |
Candidatus Micrarchaeum ARMAN-2 |
mvaD |
146329706 |
YP_001209416.1 |
Dichelobacter nodosus VCS1703A |
MPTP_0700 |
332686202 |
YP_004455976.1 |
Melissococcus plutonius ATCC 35311 |
RKLH11_3963 |
254513287 |
ZP_05125352.1 |
Rhodobacteraceae bacterium KLH11 |
Example XIV
Chemical dehydration of 3-butene-1-ol to butadiene
[0451] Alcohols can be converted to olefins by reaction with a suitable dehydration catalyst,
under appropriate conditions. Typical dehydration catalysts that convert alcohols
such as butanols and pentanols into olefins include various acid treated and untreated
alumina (e.g., γ-alumina) and silica catalysts and clays including zeolites (e.g.,
β-type zeolites, ZSM-5 or Y-type zeolites, fluoride-treated β-zeolite catalysts, fluoride-treated
clay catalysts, etc.), sulfonic acid resins (e.g., sulfonated styrenic resins such
as Amberlyst® 15), strong acids such was phosphoric acid and sulfuric acid, Lewis
acids such boron trifluoride and aluminium trichioride, many different types of metal
salts including metal oxides (e.g., zirconium oxide or titanium dioxide) and metal
chlorides (e.g.,
Latshaw B E, Dehydration of Isobutanol to Isobutylene in a Slurry Reactor, Department
of Energy Topical Report, February 1994).
[0452] Dehydration reactions can be carried out in both gas and liquid phases with both
heterogeneous and homogeneous catalyst systems in many different reactor configurations.
Typically, the catalysts used are stable to the water that is generated by the reaction.
The water is usually removed from the reaction zone with the product. The resulting
alkene(s) either exist the reactor in the gas or liquid phase (e.g., depending upon
the reactor conditions) and are captured by a downstream, purification process or
are further converted in the reactor to other compounds (such as butadiene or isoprene)
as described herein. The water generated by the dehydration reaction exits the reactor
with unreacted alcohol and alkene product(s) and is separated by distillation or phase
separation. Because water is generated in large quantities in the dehydration step,
the dehydration catalysts used are generally tolerant to water and a process for removing
the water from substrate and product may be part of any process that contains a dehydration
step. For this reason, it is possible to use wet (i.e., up to about 95% or 98% water
by weight) alcohol as a substrate for a dehydration reaction and remove this water
with the water generated by the dehydration reaction (e.g., using a zeolite catalyst
as described
U.S. Pat. Nos. 4,698,452 and
4,873,392). Additionally, neutral alumina and zeolites will dehydrate alcohols to alkenes but
generally at higher temperatures and pressures than the acidic versions of these catalysts.
[0453] Dehydration of 3-buten-1-ol to butadiene is well known in the art (
Gustav. Egloff and George. Hulla, Chem. Rev., 1945, 36 (1), pp 63-141). For example, 3-buten-1-ol is formed as an intermediate in the dehydration of 1,4-butanediol
to 1,3-butadiene (
Sato, et al, Catalysis Communications, 5 (8), 2004, p. 397-400).
Example XV
Pathways to 2,4-pentadienoate, 3-butene-1-ol and 1,3-butadiene
[0454] This example describes pathways to 2,4-pentadienoate, 3-butene-1-ol and 1,3-butadiene.
Novel pathways to the intermediates 3HP4, 3,5-dihydroxypentanoate any 3-butene-1-ol
are shown in Figures 18-21. Pathways to 3HP4 are shown in Figures 18, 19 and 20 (new).
Pathways to 3,5-dihydroxypentanoate and 3-butene-1-ol are shown in Figures 19 and
21 (new). Additional pathways to the butadiene precursor 2,4-pentadienoate are shown
in Figures 19,20 and 21 (new).
[0455] Several pathways to 2,4-pentadienoate are disclosed in earlier examples (see for
example, Figures 4, 12, 13 14 and 15. Hydration of 2,4-pentadienoate yields 3-hydroxypent-4-enoate
as shown in Figure 18 (new). 3-Hydroxypent-4-enoate can be subsequently decarboxylated
to butadiene by a 3-hydroxyacid decarboxylase, also described above.
[0456] Additional pathways from acrylyl-CoA and 3-HP-CoA to 2,4-pentadienoate are shown
in Figure 19. Also shown here are pathways to 3HP4, 3-butene-1-ol and butadiene.
[0457] Pathways to 3HP4 from acrylyl-CoA include: steps M/O/T, steps M/N/T
[0458] Pathways to 3HP4 from 3-HP-CoA include: steps A/L/O/P, steps A/L/N/T
[0459] Pathways to 3HP4 and 24PD from succinyl-CoA are shown in Figure 20. Succinyl-CoA
and acetyl-CoA are first joined by 3-oxoadipyl-CoA thiolase to form 3-oxoadipyl-CoA
(Step A). In one pathway the 3-oxo group of 3-oxoadipyl-CoA is reduced to form 3-hydroxyadipyl-CoA
(Step G). The CoA moiety is then converted to an acid group by a CoA hydrolase, synthetase,
or transferase. 3-Hydroxyadipate is then oxidized to form 3-hydroxyhex-4-enedioate
(Step I). This product is then decarboxylated to form 3HP4. In an alternate route,
3-oxoadipyl-CoA is converted to 3-oxoadipate by a CoA transferase, synthetase or hydrolase
in Step B. 3-Oxoadipate is then reduced to 3-hydroxyadipate (Step K), which is converted
to 3HP4 as previously described. Alternately, 3-oxoadipate is converted to 2-fumarylacetate
in Step C. The 3-oxo group of 2-fumarylacetate is then reduced to 3-hydroxyhex-4-enedioate,
which is decarboxylated to 3HP4. In yet another embodiment, the 2-fumarylacetate is
decarboxylated to form 3-oxopent-4-enoate (Step D), which is subsequently reduced
to 3HP4 (Step E). 3HP4 can then be converted to butadiene in one step by decarboxylation
by a 3HP4 decarboxylase (Step M), or in two steps by dehydration to 2,4-pentadiene
followed by decarboxylation (Steps F, N).
[0460] Pathways to 3-butene-1-ol, butadiene and 2,4-pentadienoate from malonyl-CoA and acetyl-CoA
are shown in Figure 21. In pathways, malonyl-CoA and acetyl-CoA are joined by a thiolase
to form 3-oxoglutaryl-CoA. This intermediate can then be converted to 3,5-dihydroxypentanoate
by several alternate routes (Steps B/C/D, Steps B/G, Steps H/I/J, Steps H/L/D, K/J).
Once formed, 3,5-dihydroxypentanoate can be decarboxylated by a 3-hydroxyacid decarboxylase
to 3-butene-1-ol (Step M). Subsequent dehydration by a 3-butene-1-ol dehydrogenase
or a chemical catalyst yields butadiene (Step O). Alternately, 3,5-dihydroxypentanoate
can be dehydrated to 5-hydroxypent-2-enoate as shown in Step E. This intermediate
can then be decarboxylated to 3-butene-1-ol (Step N) or further dehydrated to 2,4-pentadienoate
(Step F).
[0461] Enzymes for catalyzing the transformations shown in Figures 18-21 are categorized
by EC number (Table 1) and described further below.
Label |
Function |
Step |
|
|
19 I,B,N,P |
|
|
20 G,K,L,E |
1.1.1.a |
Oxidoreductase (oxo to alcohol) |
21 B,D,J,I,L |
1.1.1.c |
Oxidoreductase (2 step, acyl-CoA to alcohol) |
21 G,K |
1.2.1.b |
Oxidoreductase (acyl-CoA to aldehyde) |
21 C,H |
1.3.1.a |
Oxidoreductase (alkene to alkane) |
20 I,C |
|
|
19A,M |
2.3.1.b |
Beta-ketothiolase |
20A,21A |
|
|
19F,O,G,T,E,H |
2.8.3.a |
Coenzyme-A transferase |
20B,H |
|
|
19F,O,G,T,E,H |
3.1.2.a |
Thiolester hydrolase (CoA specific) |
20 B,H |
|
|
19 U,Y,V,X |
|
|
20 D,J,M,N |
4.1.1.a |
Carboxy-lyase |
21 M,N,P |
|
|
19 S,K,L,R,D,C,J,Q,W |
|
|
20 F |
4.2.1.a |
Hydro-lyase |
21 E,F,O |
|
|
19F,O,G,T,E,H |
6.2.1.a |
Acid-thiol ligase |
20 B,H |
[0462] Several reactions shown in Figures 19-21 are catalyzed by alcohol dehydrogenase enzymes.
These reactions include Steps B, I, N and P of Figure 19, Steps E, G, K and L of Figure
20 and Steps B, D, I, J and L of Figure 21.
[0463] Exemplary genes encoding enzymes that catalyze the reduction of an aldehyde to alcohol
(i.e., alcohol dehydrogenase or equivalently aldehyde reductase) include
alrA encoding a medium-chain alcohol dehydrogenase for C2-C14 (
Tani et al., Appl.Environ.Microbiol. 66:5231-5235 (2000)),
yqhd and
fucO from
E. coli (Sulzenbacher et al., 342:489-502 (2004)), and
bdh I and
bdh II from
C. acetobutylicum which converts butyryaldehyde into butanol (Walter et al., 174:7149-7158 (1992)),
YqhD catalyzes the reduction of a wide range of aldehydes using NADPH as the cofactor,
with a preference for chain lengths longer than C(3) (Sulzenbacher et al., 342:489-502
(2004);
Perez et al., J Biol.Chem. 283:7346-7353 (2008)). The
adhA gene product from
Zymomonas mobilisE has been demonstrated to have activity on a number of aldehydes including formaldehyde,
acetaldehyde, propionaldehyde, butyraldehyde, and acrolein (
Kinoshita et al., Appl Microbiol Biotechnol 22:249-254 (1985)). Additional aldehyde reductase candidates are encoded by
bdh in
C. saccharoperbutylacetonicum and
Cbei_1722, Cbei_2181 and
Cbei_2421 in
C. Beijerinckii. Additional aldehyde reductase gene candidates in
Saccharomyces cerevisiae include the aldehyde reductase GRE3, ALD2-6 and HFD1, glyoxylate reductase GOR1 and
YPL113C and glycerol dehydrogenase GCY1 (
WO 2011/022651A1;
Atsumi et al., Nature 45.1:86-89 (2008)). The enzyme candidates described previously for catalyzing the reduction of methylglyoxal
to acetol or lactaldehyde are also suitable lactaldehyde reductase enzyme candidates.
Protein |
GENBANK ID |
GI NUMBER |
ORGANISM |
alrA |
BAB12273.1 |
9967138 |
Acinetobacter sp. strain M=1 |
ADH2 |
NP_014032.1 |
6323961 |
Saccharomyces cerevisiae |
yqhD |
NP_417484.1 |
16130909 |
Escherichia coli |
fucO |
NP_417279.1 |
16130706 |
Escherichia coli |
bdh I |
NP_149892.1 |
15896543 |
Clostridium acetobutylicum |
bdh II |
NP_349891.1 |
15896542 |
Clostridium acetobutylcum |
adhA |
YP_ 162971.1 |
56552132 |
Zymomonas mobilis |
bdh |
BAF45463.1 |
124221917 |
Clostridium saccharoperbutylacetonicum |
Cbei_1722 |
YP_001308850 |
150016596 |
Clostridium beijerinckii |
Cbei_2181 |
YP_001309304 |
150017050 |
Clostridium beijerinckii |
Cbei_2421 |
YP_001309535 |
150017281 |
Clostridium beijerinckii |
GRE3 |
P38715.1 |
731691 |
Saccharomyces cerevisiae |
ALD2 |
CAA89806.1 |
825575 |
Saccharomyces cerevisiae |
ALD3 |
NP_013892.1 |
6323821 |
Saccharomyces cerevisiae |
ALD4 |
NP_015019.1 |
6324950 |
Saccharomyces cerevisiae |
ALD5 |
NP_010996.2 |
330443526 |
Saccharomyces cerevisiae |
ALD6 |
ABX39192.1 |
160415767 |
Saccharomyces cerevisiae |
HFD1 |
Q04458.1 |
2494079 |
Saccharomyces cerevisiae |
GOR1 |
NP_014125.1 |
6324055 |
Saccharomyces cerevisiae |
YPL113C |
AAB68248.1 |
1163100 |
Saccharomyces cerevisiae |
GCY1 |
CAA99318.1 |
1420317 |
Saccharomyces cerevisiae |
[0465] Another exemplary aldehyde reductase is methylmalonate semialdehyde reductase, also
known as 3-hydroxyisobutyrate dehydrogenase (EC 1.1.1.31). This enzyme participates
in valine, leucine and isoleucine degradation and has been identified in bacteria,
eukaryotes, and mammals. The enzyme encoded by
P84067 from
Thermus thermophilus HB8 has been structurally characterized (
Lokanath et al., J Miol Biol, 352:905-17 (2005)). The reversibility of the human 3-hydroxyisobutyrate dehydrogenase was demonstrated
using isotopically-labeled substrate (
Manning et al., Biochem, J, 231:481-4 (1985)). Additional genes encoding this enzyme include
3hidh in
Homo sapiens (
Hawes et al., Methods Enzymol, 324:218-228 (2000)) and
Oryctolagus cuniculus (Hawes et al.,
supra;
Chowdhury et al., Biosci.Biotechnol Biochem. 60:2043-2047 (1996)),
mmsB in
Pseudomonas aeruginosa and
Pseudomonas putida, and
dhat in
Pseudomonas putida (
Aberhart et al., J Chem. Soc.[Perkin 1] 6:1404-1406 (1979);
Chowdhury et al., Biosci.Biotechnol Biochem. 60:2043-2047 (1996);
Chowdhury et al., Biosci.Biotechnol Biochem, 67:438-441 (2003)). Several 3-hydroxylisobutyrate dehydrogenase enzymes have been characterized in
the reductive direction, including
mmsb from
Pseudomonas aeruginosa (
Gokarn et al., US Patent 739676, (2008)) and
mmsB from
Pseudomonas putida.
PROTEIN |
GENBANK ID |
GI NUMBER |
ORGANISM |
P84067 |
P84067 |
75345323 |
Thermus thermophilus |
3hidh |
P31937.2 |
12643395 |
Homo sapiens |
3hidh |
P32185.1 |
416872 |
Oryctolagus cuniculus |
mmsB |
NP_746775.1 |
26991350 |
Pseudomonas putida |
mmsB |
P28811.1 |
127211 |
Pseudomonas aeruginosa |
dhat |
Q59477.1 |
2842618 |
Pseudomonas putida |
[0466] There exist several exemplary alcohol dehydrogenases that reduce a ketone to a hydroxyl
functional group. Two such enzymes from
E. coli are encoded by malate dehydrogenase (
mdh) and lactate dehydrogenase (
ldhA). In addition, lactate dehydrogenase from
Ralstonia eutropha has been shown to demonstrate activities on 2-ketoacids of various chain lengths
includings lactate, 2-oxobutyrate, 2-oxopentanoate and 2-oxoglatarate (
Stembuchel et al., Eur.J.Biochem. 130:329-334 (1983)). Conversion of alpha-ketoadipate into alpha-hydroxyadipate can be catalyzed by
2-ketoadipate reductase, an enzyme reported to be found in rat and in human placenta
(
Suda et al., Arch.Biochem.Biophys. 176:610-620 (1976);
Suda et al., Biochem.Biophys.Res.Commun. 77.586-591 (1977)). An additional oxidoreductase is the mitochondrial 3-hydroxybutyrate dehydrogenase
(
bdh) from the human heart which has been cloned and characterized (
Marks et al., J.Biol.Chem. 267:15459-15463 (1992)). Alcohol dehydrogenase enzymes of
C. beijerinckii (
Ismaiel et al., J.Bacteriol. 175:5097-5105 (1993)) and
T. brockii (
Lamed et al., Biochem.J. 195.183-190 (1981);
Peretz et al., Biochemistry. 28:6549-6555 (1989)) convert acetone to isopropanol. Methyl ethyl ketone reductase catalyzes the reduction
of MEK to 2-butanol. Exemplary MEK reductase enzymes can be found in
Rhoclococcus ruber (
Kosjek et al., Biotechnol Bioeng. 86:55-62 (2004)) and
Pyrococcus furiousus (
van der et al., Eur.J.Biochem. 268:3062-3068 (2001)).
Protein |
GenBank Accession No. |
GI No. |
Organism |
mdh |
AAC76268.1 |
1789632 |
Escherichia coli |
ldhA |
NP_415898.1 |
16129341 |
Escherichia coli |
ldh |
YP_725182.1 |
113866693 |
Ralstonia eutropha |
bdh |
AAA58352.1 |
177198 |
Homo sapiens |
adh |
AAA23199.2 |
60592974 |
Clostridium beijerinckii NRRL B593 |
adh |
P14941.1 |
113443 |
Thermoanaerobacter brockii HTD4 |
sadh |
CAD36475 |
21615553 |
Rhodococcus ruber |
adhA |
AAC25556 |
3288810 |
Pyrococcus furiosus |
[0467] A number of organisms can catalyze the reduction of 4-hydroxy-2-butanone to 1,3-butanediol,
including those belonging to the genus
Bacillus, Brevibacterium, Candida, and
Klebsiella among others, as described by Matsuyama et al. ((1995)). A mutated
Rhodococcus phenylacetaldehyde reductase (Sar268) and a
Leifonia, alcohol dehydrogenase have also been shown to catalyze this transformation at high
yields (
Itoh et al., Appl.Microbiol Biotechnol. 75:1249-1256 (2007)).
[0468] Alcohol dehydrogenase enzymes that reduce 3-oxoacyl-CoA substrates to their corresponding
3-hyroxyacyl-CoA product are also relevant to the pathways depicted in Figures 19-21.
3-Oxoacyl-CoA dehydrogenase enzymes (EC 1.1.1.35) convert 3-oxoacyl-CoA molecules
into 3-hydroxyacyl-CoA molecules and are often involved in fatty acid beta-oxidation
or phenylacetate catabolism. For example, subunits of two fatty acid oxidation complexes
in
E. coli, encoded by
fadB and
fadJ, function as 3-hydroxyacyl-CoA dehydrogenases (
Binstock et al., Methods Enzymol. 71 Pt C:403-411 (1981)). Given the proximity in
E. coli of
paaH to other genes in the phenylacetate degradation operon (Nogales et al., 153:357-365
(2007)) and the fact that
paaH mutants cannot grow on phenylacetate (
Ismail et al., Eur.J Biochem. 270:3047-3054 (2003)), it is expected that the
E. coli paaH gene also encodes a 3-hydroxyacyl-CoA dehydrogenase. Acetoacetyl-CoA reductase participates
in the acetyl-CoA fermentation pathway to butyrate in several species of
Clostridia and has been studied in detail (
Jones et al., Microbiol Rev. 50:484-524 (1986)). The enzyme from
Clostridium acetobutylicum, encoded by
hbd, has been cloned and functionally expressed in
E. coli (
Youngleson et al., J Bacteriol. 171:6800-6807 (1989)). Yet other genes demonstrated to reduce acetoacetyl-CoA to 3-hydroxybutyryl-CoA
are
phbB from
Zoogloea ramigera (
Ploux et al., Eur.J Biochem. 174:177-182 (1988)) and
phaB from
Rhodobacter sphaeroides (
Alber et al., Mol.Microbiol 61:297-309 (2006)). The former gene is NADPH-dependent, its nucleotide sequence has been determined
(
Peoples et al., Mol.Microbiol 3:349-357 (1989)) and the gene has been expressed in
E. coli. Substrate specificity studies on the gene led to the conclusion that it could accept
3-oxopropionyl-CoA as a substrate besides acetoacetyl-CoA (
Ploux et al., Eur.J Biochem. 174:177-182 (1988)). Additional genes include
phaB in
Paracoccus denitrificans, Hbd1 (C-terminal domain) and
Hbd2 (N-terminal domain) in
Clostridium kluyveri (
Hillmer and Gottschalk, Biochim. Biophys. Acta 3334:12-23 (1974)) and
HSD17B10 in
Bos taurus (Wakil et al., J Biol.Chem. 207:631-638 (1954)). The enzyme from
Paracoccus denitrificans has been functionally expressed and characterized in
E. coli (
Yabutani et al., FEMS Microbiol Lett. 133:85-90 (1995)). A number of similar enzymes have been found in other species of
Clostridia and in
Metallosphaera sedula (
Berg et al., Science. 318:1782-1786 (2007)). The enzyme from
Candida tropicalis is a component of the peroxisomal fatty acid beta-oxidation multifunctional enzyme
type 2 (MFE-2). The dehydrogenase B domain of this protein is catalytically active
on acetoacetyl-CoA. The domain has been functionally expressed in
E. coli, a crystal structure is available, and the catalytic mechanism is well-understood
(
Ylianttila et al., Biochem Biophys Res Commun 324:25-30 (2004);
Ylianttila et al., J Mol Biol 358:1286-1295 (2006)).
Protein |
GENBANK ID |
GI NUMBER |
ORGANISM |
fadB |
P21177.2 |
119811 |
Escherichia coli |
fadJ |
P77399,1 |
3334437 |
Escherichia coli |
paaH |
NP_415913.1 |
16129356 |
Escherichia coli |
Hbd2 |
EDK34807.1 |
146348271 |
Clostridium kluyveri |
Hbd1 |
EDK32512,1 |
146345976 |
Clostridium kluyveri |
HSD17B10 |
O02691.3 |
3183024 |
Bos taurus |
phbB |
P23238.1 |
130017 |
Zoogloea ramigera |
phaB |
YP_353825.1 |
77464321 |
Rhodobacter sphaeroides |
phaB |
BAA08358 |
675524 |
Paracoccus denitrificans |
Hbd |
NP_349314.1 |
15895965 |
Clostridium acetobutylicum |
Hbd |
AAM14586.1 |
20162442 |
Clostridium beijerinckii |
Msed_1423 |
YP_001191505 |
146304189 |
Metallosphaera sedula |
Msed_0399 |
YP_01190500 |
146303184 |
Metallosphaera sedula |
Msed_0389 |
YP_001190490 |
146303174 |
Metallosphaera sedula |
Msed_1993 |
YP_001192057 |
146304741 |
Metallosphaera sedula |
Fox2 |
Q02207 |
399508 |
Candida tropicalis |
[0469] Bifunctional oxidoreductases convert an acyl-CoA to its corresponding alcohol. Enzymes
with this activity are required to convert 3-hydroxyglutaryl-CoA to 3,5-dihydroxypentanoate
(Figure 21, Step G) and 3-oxoglutaryl-CoA to 5-hydroxy-3-oxopentanoate (Figure 21,
Step K).
[0470] Exemplary bifunctional oxidoreductases that convert an acyl-CoA to alcohol include
those that transform substrates such as acetyl-CoA to (ethanol (e.g.,
adhE from
E. coli (
Kessler et al., FERS.Lett. 281:59-63 (1991))) and butyryl-CoA to butanol (e.g.
adhE2 from
C. acetobutylicum (
Fontaine et al., J.Bacteriol. 184:821-830 (2002))). The
C. acetobutylicum enzymes encoded by
bdh I and
bdh II (
Walter, et al., J. Bacteriol. 174:7149-7158 (1992)), reduce acetyl-CoA and butyryl-CoA to ethanol and butanol, respectively. In addition
to reducing acetyl-CoA to ethanol, the enzyme encoded by
adhE in
Leuconostoc mesenteroides has been shown to oxide the branched chain compound isobutyraldehyde to isobutyryl-CoA
(
Kazahaya et al., J.Gen.Appl.Microbiol. 18:43-55 (1972);
Koo et al., Biotechnol Lett, 27:505-510 (2005)). Another exemplary enzyme can convert malonyl-CoA to 3-HP. An NADPH-dependent enzyme
with this activity has characterized in
Chloroflexux aurantiacus where it participates in the 3-hydroxypropionate cycle (
Hugler et al., J Bacteriol, 184:2404-2410 (2002);
Strauss et al., Eur J Biochem, 215:633-643 (1993)). This enzyme, with a mass of 300 kDa, is highly substrate-specific and shows little
sequence similarity to other known oxidoreductases (Hugler et al.,
supra). No enzymes in other organism have been shown to catalyze this specific reaction;
however there is bioinformatic evidence that other organism may have similar pathways
(
Klatt et al., Env Microbiol, 9:2067-2078 (2007)). Enzyme candidates in other organisms including
Roseiflexus castenholzii, Erythrobacter sp. NAP1 and marine gamma proteobacterium HTCC2080 can be inferred by sequence similarity.
Protein |
GenBank ID |
GI Number |
Oraganism |
adhE |
NP_415757.1 |
16129202 |
Escherichia coli |
adhE2 |
AAK09379.1 |
12958626 |
Clostridium acetobutylicum |
bdh I |
NP_349892.1 |
15896543 |
Clostridium acetobutylicum |
bdh II |
NP_349891.1 |
15896542 |
Clostridium acetobutylicum |
Protein |
GenBank ID |
GI Number |
Organism |
adhE |
AAV66076.1 |
55818563 |
Leuconostoc mesenteroides |
mcr |
AAS20429.1 |
42561982 |
Chloroflexus aurantiacus |
Rcas_2929 |
YP_001433009.1 |
156742880 |
Roseiflexus castenholzii |
N4P1_02720 |
ZP_01039179.1 |
85708113 |
Erythrobacter sp. NAP1 |
MGP2080_00535 |
ZP_01626393.1 |
119504313 |
marine gamma proteobacterium HTCC2080 |
[0471] Longer chain acyl-CoA molecules can be reduced to their corresponding alcohols by
enzymes such as the jojoba (
Simmondsia chinensis)
FAR which encodes an alcohol-forming fatty acyl-CoA reductase. Its overexpression in
E. coli resulted in FAR. activity and the accumulation of fatty alcohol (
Metz et al., Plant Physiol, 122:635-644 (2000)).
Protein |
GenBank ID |
GI Number |
Organism |
FAR |
AAD38039.1 |
5020215 |
Simmondsia chinensis |
[0472] Another candidate for catalyzing these steps is 3-hydroxy-3-methylglutaryl-CoA reductase
(or HMG-CoA reductase). This enzyme naturally reduces the CoA group in 3-hydroxy-3-methylglutaryl-CoA
to an alcohol forming mevalonate. The
hmgA gene of Sulfolobus
solfataricus, encoding 3-hydroxy-3-methylglutaryl-CoA reductase, has been cloned, sequenced, and
expressed in
E. coli (
Bochar et al., J Bacteriol. 179:3632-3638 (1997)).
S.
cerevisiae also has two HMC-CoA reductases in it (
Basson et al., Proc.Natl.Acad.Sci.U.S.A 83:5563-5567 (1986)). The gene has also been isolated from
Arabidopsis thaliana and has been shown to complement the HMG-COA reductase activity in
S. cerevisiae (
Learned et al., Proc.Natl.Acad.Sci.U.S.A 86:2779-2783 (1989)).
Protein |
GenBank ID |
GI Number |
Orgnism |
HMG1 |
CAA86503.1 |
587536 |
Saccharomyces cerevisiae |
HMG2 |
NP_013555 |
6323483 |
Saccharomyces cerevisiae |
HMG1 |
CAA70691.1 |
1694976 |
Arabidopsis thaliana |
hmgA |
AAC45370.1 |
2130564 |
Sulfolobus solfataricus |
Acyl-CoA reductase in the 1.2.1 family reduce an acyl-CoA to its corresponding aldehyde.
Such a conversion is required in steps C and H of Figure 21. Several acyl-CoA dehydrogenase
enzymes have been described in the open literature and represent suitable candidates
for these steps. These are described below.
[0473] Exemplary acyl-CoA reductase enzymes include fatty acyl-CoA reductase, succinyl-CoA
reductase (EC 1.2.1.76), acetyl-CoA reductase and butyryl-CoA reductase. Exemplary
fatty acyl-CoA reductase enzymes are encoded by
acr1 of
Acinetobacter calcoaceticus (
Reiser, Journal of Bacteriology 179:2969-2975 (1997)) and
Acinetobacter sp.
M-
1 (
Ishige et al., Appl. Environ. Microbiol. 68:1192-1195 (2002)). Enzymes with succinyl-CoA reductase activity are encoded by
sucD of
Clostridium kluyveri (
Sohling, J. Bacteriol. 178:871-880 (1996)) and
sucD of
P.
gingivalis (
Takahashi, J. Bacteriol 182:4704-4710 (2000)). Additional succinyl-CoA reductase enzymes participate in the 3-hydroxypropionate/4-hydroxybutyrate
cycle of thermophilic archaea including
Metallosphaera sedula (
Berg et al., Science 318:1782-1786 (2007)) and
Thermoproteus neutrophilus (
Ramos-Vera et al., J Bacteriol., 191:4286-4297 (2009)). The
M. sedula enzyme, encoded by
Msed_0709, is strictly NADPH-dependent and also has malonyl-CoA reductase activity. The
T. neutrophilus enzyme is active with both NADPH and NADH. The enzyme acylating acetaldehyde dehydrogenase
in
Pseudomonas sp, encoded by
bphG, is yet another as it has been demonstrated to oxidize and acylate acetaldehyde, propionaldehyde,
butyraldehyde, isobutyraldehyde and formaldehyde (
Powlowski, J. Bacteriol. 175:377-385 (1993)). In addition to reducing acetyl-CoA to ethanol, the enzyme encoded by
adhE in
Leuconostoc mesenteroides has been shown to oxidize the branched chain compound isobutyraldehyde to isobutyryl-CoA
(
Kazahaya, J. Gen. Appl. Microbiol. 18:43-55 (1972); and
Koo et al., Biotechnol Lett. 27:505-510 (2005)). Butyraldehyde dehydrogenase catalyzes a similar reaction, conversion of butyryl-CoA
to butyraldehyde, in solventogenic organisms such as
Clostridium saccharoperbutylacetonicum (
Kosaka et al., Biosci Biotechnol Biochem., 71:58-68 (2007)).
Protein |
GenBank ID |
GI Number |
Organism |
acrI |
YP_047869.1 |
50086359 |
Acinetobacter calcoaceticus |
acrI |
AAC45217 |
1684886 |
Acinetobacter baylyi |
acrI |
BAB85476.1 |
18857901 |
Acinetobacter sp. Strain M-I |
MSED_0709 |
YP_001190808.1 |
146303492 |
Metallosphaera sedula |
Tneu_0421 |
ACB39369.1 |
170934108 |
Thermoproteus neutrophilus |
sucD |
P38947.1 |
172046062 |
Clostridium kluyveri |
sucD |
NP_904963.1 |
34540484 |
Porphyromonas gingivalis |
bphG |
BAA03892.1 |
425213 |
Pseudomonas sp |
adhE |
AAV66076.1 |
55818563 |
Leuconostoc mesenteroides |
Bld |
AAP42563.1 |
31075383 |
Clostridium saccharoperbutylacetonicum |
[0474] An additional enzyme type that converts an acyl-CoA to its corresponding aldehyde
is malonyl-CoA reductase which transforms malonyl-CoA to malonic semialdehyde. Malonyl-CoA
reductase is a key enzyme in autotrophic carbon fixation via the 3-hydroxypropionate
cycle in thermoacidophilic archaeal bacteria (
Berg, Science 318:1782-1786 (2007); and
Thauer, Science 318:1732-1733 (2007)). The enzyme utilizes NADPH as a cofactor and has been characterized in
Metallosphaera and
Sulfolobus sp. (
Alber et al., J. Bacteriol. 188:8551-8559 (2006); and
Hugler, J. Bacteriol. 184:2404-2410 (2002)). The enzyme is encoded by
Msed_0709 in
Metallosphaera sedula (
Alber et al., J. Bacteriol. 188:8551-8559 (2006); and
Berg, Science 318:1782-1786 (2007)). A gene encoding a malonyl-CoA reductase from
Sulfolobus tokodaii was cloned and heterotogously expressed in
E.
coli (
Alber et al., J. Bacteriol 188:8551-8559 (2006), This enzyme has also been shown to catalyze the conversion of methylmalonyl-CoA
to its corresponding aldehyde (
WO2007141208 (2007)). Although the aldehyde dehydrogenase functionality of these enzymes is similar
to the bifunctional dehydrogenase from
Chloroflexus aurantiacus, there is little sequence similarity. Both malonyl-CoA reductase enzyme candidates
have high sequence similarity to aspartate-semialdehyde dehydrogenase, an enzyme catalyzing
the reduction and concurrent dephosphorylation of aspartyl-4-phosphate to aspartate
semialdehyde. Additional gene candidates can be found by sequence homology to proteins
in other organisms including
Sulfolobus solfataricus and
Sulfolobus acidocaldarius and have been listed below. Yet another candidate for CoA-acytating aldehyde dehydrogenase
is the
ald gene from
Cloctridium beijerinckii (
Toth, Appl. Environ. Microbiol. 65:4973-4980 (1999). This enzyme has been reported to reduce acetyl-CoA and butyryl-CoA to their corresponding
aldehydes. This gene is very similar to
eutE that encodes acetaldehyde dehydrogenase of
Salmonella typhimurium and
E.
coli (
Toth, Appl. Environ. Microbiol. 65:4973-4980 (1999).
Protein |
GenBank ID |
GI Number |
Organism |
Msed_0709 |
YP_001190808.1 |
146303492 |
Metallosphaera sedula |
Mcr |
NP_378167.1 |
15922498 |
Sulfolobus tokodaii |
asd-2 |
NP_343563.1 |
15898958 |
Sulfolobus solfataricus |
Saci_2370 |
YP_256941.1 |
70608071 |
Sulfolobus acidocaldarius |
Ald |
AAT66436 |
49473535 |
Clostridium beijerinckii |
eutE |
AAA80209 |
687645 |
Salmonella typhimurium |
eutE |
P77445 |
2498347 |
Escherichia coli |
[0475] 2-Enoate reductase enzymes, some of which, are reversible, are known to catalyze
the NAD(P)H-dependent reduction of a wide variety of α, β-unsaturated carboxylic acids
and aldehydes(Rohdich et al., 276:5779-5787 (2001)). These enzymes represent suitable
candidates to carry out the transformations depicted by steps I and C of Figure 20.
Several examples are provided below.
[0476] In the recently published genome sequence of
C. kluyveri, 9 coding sequences for enoate reductases were reported, out of which one has been
characterized (
Seedorf et al., Proc.Natl. Acad.Sci U.S.A 105:2128-2133 (2008)). The
enr genes from both
C. tyrobutyricum and
M.
thermoaceticum have been cloned and sequenced and show 59% identity to each other. The former gene
is also found to have approximately 75% similarity to the characterized gene in
C kluyveri (Giesel et al., 135:51-57 (1983)), It has been reported based on these sequence results
that
enr is very similar to the dienoyl CoA reductase in
E. coli (
fadH) (
Rohdich et al., J Biol.Chem. 276:5779-5787 (2001)). The
C.
thermoaceticum enr gene has also been expressed in a catalytically active form in
E. coli (
Rohdich et al., J Biol. Chem. 276:5779-5787 (2001)). This enzyme exhibits activity on a broad range of alpha, beta-unsaturated carbonyl
compounds.
Protein |
GenBank ID |
GI Number |
Organism |
enr |
ACA54153.1 |
169405742 |
Clostridium botulinum A3 str |
enr |
CAA71086.1 |
2765041 |
Clostridium tyrobutyricum |
enr |
CAA76083.1 |
3402834 |
Clostridium kluyveri |
enr |
YP_430895.1 |
83590886 |
Moorella thermoacetica |
fadH |
NP_417552.1 |
16130976 |
Escherichia coli |
[0477] Another candidate 2-enoate reductase is maleylacetate reductase (MAR, EC 1.3.1.32),
an enzyme catalyzing the reduction of 2-maleylacetate (4-oxohex-2-enedioate) to 3-oxoadipate.
MAR enzymes naturally participate in aromatic degradation pathways (
Kaschabek et al., J Bacteriol. 175:6075-6081 (1993);
Kaschabek et al., J Bacteriol. 177:320-325 (1995);
Camara et al., J Bacteriol. (2009);
Huang et al., Appl Environ. Microbiol 72:7238-7245 (2006)). The enzyme activity was identified and characterized in
Pseudomonas sp. strain B13 (Kaschabek et al., 175:6075-6081 (1993); Kaschabek et al., 177:320-325
(1995)), and the coding gene was cloned and sequenced (
Kasberg et al., J Bacteriol 179:3801-3803 (1997)). Additional MAR gene candidates include
clcE gene from
Pseudomonas sp. strain B13 (
Kasberg et al., J Bacteriol. 179:3801-3803 (1997)),
macA gene from
Rhodococcus opacus (Seibert et al., 180:3503-3508 (1998)), the
macA gene from
Ralstonia eutropha (also known as
Cupriavidusnecator) (
Seibert et al., Microbiology 150:463-472 (2004)),
tfdFII from
Ralstonia eutropha (
Seibert et al., J Bacteriol. 175:6745-6754 (1993)) and
NCgl1112 in
Corynebacterium glutamicum (
Huang et al., Appl Environ. Microbiol 72:7238-7245 (2006)). A MAR in
Pseudomonas reinekei MT1, encoded by
ccaD, was recently identified (
Camara et al., J Bacteriol. (2009)).
Protein |
GenBank ID |
GI Number |
Organism |
cleE |
3913241 |
O30847.1 |
Pseudomonas sp. strain B 13 |
macA |
7387876 |
O84992.1 |
Rhodococcus opacus |
macA |
5916089 |
AAD55886 |
Cupriavidus necator |
tfdFII |
1747424 |
AAC44727.1 |
Ralstonia eutropha JMP134 |
NCgl1112 |
19552383 |
NP_600385 |
Corynebacterium glutamicum |
ccaD |
134133940 |
ABO61029.1 |
Pseudomonas reinekei MT1 |
[0478] Step A of Figures 19-21 and Step M of Figures 19 require condensation of either 3-hydroxypropionyl-CoA,
acrylyl-CoA, succinyl-CoA or malonyl-CoA with acetyl-CoA. Several -ketothiolase enzymes
have been described in the open literature and represent suitable candidates for these
steps. These are described below.
[0479] For example, 3-Oxoadipyl-CoA thiolase represents one type of beta-ketothiolase enzyme
that is suitable for the aforementioned steps. 3-Oxoadipyl-CoA thiolase (EC 2.3.1.174)
naturally converts beta-ketoadipyl-CoA to succinyl-CoA and acetyl-CoA and is a key
enzyme of the beta-ketoadipate pathway for aromatic compound degradation. The enzyme
is widespread in soil bacteria and fungi including
Pseudomonas putida (
Harwood et al., J Bacteriol. 176:6479-6488 (1994)) and
Acinetobacter calcoaceticus (
Doten et al., J Bacteriol. 169:3168-3174 (1987)). The gene products encoded by
pcaF in
Pseudomonas strain B13 (
Kaschabek et al., J Bacteriol. 184:207-215 (2002)),
phaD in
Pseudomonas putida U (
Olivera et al., Proc. Natl.Acad. Sci U.S.A 95:6419-6424 (1998))
,paaE in
Pseudomonas fluorescens ST (
Di et al., Arch.Microbiol 188:17-125 (2007)), and
paaJ from
E.
coli (
Nogales et al., Microbiology 153:357-365 (2007)) also catalyze this transformation. Several beta-ketothiotases exhibit significant
and selective activities in the oxoadipyl-CoA forming direction including
bkt from
Pseudomonas putida, pcaF and
bkt from
Pseudomonas aeruginosa PAO1, bkt from
Burkholderia ambifaria AMMD,
paaJ from
E. coli, and
phaD from.
P.
putida.
Protein |
GI# |
GenBank Accession # |
Organism |
paaJ |
16129358 |
NP_415915.1 |
Escherichia coli |
pcaF |
17736947 |
AAL02407 |
Pseudomonas knackmussii (B13) |
phaD |
3253200 |
AAC24332.1 |
Pseudomonas putida |
pcaF |
506695 |
AAA85138.1 |
Pseudomonas putida |
pcaF |
141777 |
AAC37148.1 |
Acinetobacter calcoaceticus |
paaE |
106636097 |
ABF82237.1 |
Pseudomonas fluorescens |
bkt |
115360515 |
YP_777652.1 |
Burkholderia ambifaria AMMD |
bkt |
9949744 |
AAG06977.1 |
Pseudomonas aeruginosa PAO1 |
pcaF |
9946065 |
AAG03617.1 |
Pseudomonas aeruginosa PAO1 |
[0480] Glutaryl-CoA and acetyl-CoA are condensed to form 3-oxopimeloyl-CoA by oxopimeloyl-CoA-glutaryl-CoA
acyltransferase, a beta-ketothiolase (EC 2.3.1.16). An enzyme catalyzing this transformation
is found in
Ralstonia eutropha (formerly known as
Alcaligenes eutrophus), encoded by genes
bktB and
bktC (
Slater et al., J.Bacteriol. 180:1979-1987 (1998); Haywood et al., 52:91-96 (1988)). The sequence of the BktB protein is known; however,
the sequence of the BktC protein has not been reported. The
pim operon
of Rhodopseudomonas palustris also encodes a beta-ketothiolase, encoded by
pimB, predicted to catalyze this transformation in the degradative direction during benzoyl-CoA
degradation (Harrison et al., 151:727-736 (2005)). A beta-ketothiolase enzyme candidate
in
S. aciditrophicus was identified, by sequence homology to
bktb (43% identity, evalue = 1e-93).
Protein |
GenBank ID |
GI Number |
Organism |
bktB |
YP_725948 |
11386745 |
Ralstonia eutropha |
pimB |
CAE29156 |
39650633 |
Rhodopseudomonas palustris |
syn_02642 |
YP_462685.1 |
85860483 |
Syntrophus aciditrophicus |
[0481] Beta-ketothiolase enzymes catalyzing the formation of beta-ketovalerate from acetyl-CoA
and propionyl-CoA may be to catalyze the condensation of acetyl-CoA with 3-hydroxypropionyl-CoA,
acrylyl-CoA, succinyl-CoA, or malonyl-CoA.
Zoogloea ramigera possesses two ketothiolases that can form -ketovaleryl-CoA from propionyl-CoA and
acetyl-CoA and
R. eutropha has a -oxidation ketothiolase that is also capable of catalyzing this transformation
(
Gruys et al., US Patent 5,958,745 (1999)). The sequences of these genes or their translated proteins have not been reported,
but several candidates in
R. eutropha, Z.
ramigera, or other organisms can be identified based on sequence homology to
bktB from
R.
eutropha. These include:
Protein |
GenBank ID |
GI Number |
Organism |
phaA |
YP_725941.1 |
113867452 |
Ralstonia eutropha |
h16_A1713 |
YP_726205.1 |
113867716 |
Ralstonia eutropha |
pcaF |
YP_728366.1 |
116694155 |
Ralstonia eutropha |
h16_B1369 |
YP_840888.1 |
116695312 |
Ralstonia eutropha |
h16_A0170 |
YP_724690.1 |
113866201 |
Ralstonia eutropha |
h16_A0462 |
YP_724980.1 |
113866491 |
Ralstonia eutropha |
h16_A1528 |
YP_726028.1 |
113867539 |
Ralstonia eutropha |
h16_B0381 |
YP_728545.1 |
116694334 |
Ralstonia eutropha |
h16_B0662 |
YP_728824.1 |
116694613 |
Ralstonia eutropha |
h16_B0759 |
YP_728921.1 |
116694710 |
Ralstonia eutropha |
h16_B0668 |
YP_728830.1 |
116694619 |
Ralstonia eutropha |
h16_A1720 |
YP_726212.1 |
113867723 |
Ralstonia eutropha |
h16_A1887 |
YP_726356.1 |
113867867 |
Ralstonia eutropha |
phbA |
P07097.4 |
135759 |
Zoogloea ramigera |
bktb |
YP_002005382.1 |
194289475 |
Cupriavidus taiwanensis |
Rmet_1362 |
YP_583514.1 |
94310304 |
Ralstonia metallidurans |
Bphy_0975 |
YP_001857210.1 |
186475740 |
Burkholderia phymatum |
[0483] Enzymes in the 2.8.3 family catalyze the reversible transfer of a CoA moiety from
one molecule to another. Such a transformation is required by steps F, O, G, T, H,
and E of figure 19 and steps B and H of Figure 20. Several CoA transferase enzymes
have been described in the open literature and represent suitable candidates for these
steps. These are described below.
[0484] Many transferases have broad specificity and thus can utilize CoA acceptors as diverse
as acetate, succinate, propionate, butyrate, 2-methylacetoacetate, 3-ketohexanoate,
3-ketopentanoate, valerate, crotonate, 3-mercaptopropionate, propionate, vinylacetate,
butyrate, among others. For example, an enzyme from
Roseburia sp. A2-183 shown, to have butyryl-CoA:acetate:CoA transferase and propionyl-CoA:acetate:CoA
transferase, activity (
Charrier et al., Microbiology 152, 179-185 (2006)). Close homologs can be found in, for example,
Roseburia intestinalis L1-82,
Roseburia inulinivorans DSM 16841,
Eubacterium rectale ATCC 33656. Another enzyme with propionyl-CoA transferase activity can be found in
Clostridium propionicum (
Selmer et al., Eur J Biochem 269, 372-380 (2002)). This enzyme can use acetate, (R)-lactate, (S)-lactate, acrylate, and butyrate
as the CoA acceptor (
Selmer et al., Eur J Biochem 269, 372-380 (2002);
Schweiger and Buckel, FEBS Letters, 171(1) 79-84 (1984)). Close homologs can be found in, for example,
Clostridium novyi NT,
Clostridium beijerinckii NCIMB 8052, and
Clostridium botulinum C str. Eklund.
YgfH encodes a propionyl CoA:succinate CoA transferase in
E. coli (
Haller et al., Biochemistry, 39(16) 4622-4629). Close homologs can be found in, for example,
Citrobacter youngae ATCC 29220,
Salmonella enterica subsp.
arizonae serovar, and
Yersinia intermedia ATCC 29909. These proteins are identified below.
Protein |
GenBank ID |
GI Number |
Organism |
Ach1 |
AAX19660.1 |
60396828 |
Roseburia sp. A2-183 |
ROSINTL182_07121 |
ZP_04743841.2 |
257413684 |
Roseburia intestinalis L1-82 |
ROSEINA2194_03642 |
ZP_03755203.1 |
225377982 |
Roseburia inulinivorans |
EUBREC_3075 |
YP_002938937.1 |
238925420 |
Eubacterium rectale ATCC 33656 |
Pct |
CAB77207.1 |
7242549 |
Clostridium propionicum |
NT01CX_2372 |
YP_878445.1 |
118444712 |
Clostridium novyi NT |
Cbei_4543 |
YP_001311608.1 |
150019354 |
Clostridium beijerinckii |
CBC_A0889 |
ZP_02621218.1 |
168186583 |
Clotridium botulinum C str. Eklund |
ygfH |
NP_417395.1 |
16130821 |
Escherichia coli |
CIT292_04485 |
ZP_03838384.1 |
227334728 |
Citrobacter youngae ATCC 29220 |
SARI_04582 |
YP_001573497.1 |
161506385 |
Salmonella enterica subsp. arizonae serovar |
yinte0001_14430 |
ZP_04635364.1 |
238791727 |
Yersinia intermedia ATCC 29909 |
[0485] An additional candidate enzyme is the two-unit enzyme encoded by
pcaI and
pcaJ in
Pseudomonas, which has been shown to have 3-oxoadipyl-CoA/succinate, transferase activity (Kaschabek
et al.,
supra). Similar enzymes based on homology exist in
Acinetobacter sp. ADP1 (
Kowalchuk et al., Gene 146:23-30 (1994)) and
Streptomyces coelicolor. Additional exemplary succinyl-CoA:3:oxoacid-CoA transferases are present in
Helicobacter pylori (
Corthesy-Theulaz et al., J.Biol.Chem. 272:25659-25667 (1997)) and
Bacillus subtilis (
Stols et al., Protein.Expr.Purif. 53:396-403 (2007)). These proteins are identified below.
Protein |
GenBank ID |
GI Number |
Organism |
pcaI |
AAN69545.1 |
24985644 |
Pseudomonas putida |
pcaJ |
NP_746082.1 |
26990657 Pseudomonas |
putida |
pcaI |
YP_046368.1 |
50084858 |
Acinetobacter sp. ADP1 |
pcaJ |
AAC37147.1 |
141776 |
Acinetobacter sp. ADP1 |
pcaI |
NP_630776.1 |
21224997 |
Streptomyces coelicolor |
pcaJ |
NP_63 0775.1 |
21224996 |
Streptomyces coelicolor |
HPAG1_0676 |
YP_627417 |
108563101 |
Helicobacter pylori |
HPAG1_0677 |
YP_627418 |
108563102 |
Helicobacter pylori |
ScoA |
NP_391778 |
16080950 |
Bacillus subtilis |
ScoB |
NP_391777 |
16080949 |
Bacillus subtilis |
[0486] A CoA transferase that can utilize acetate as the CoA acceptor is acetoacetyl-CoA
transferase, encoded by the
E. coli atoA (alpha, subunit) and
atoD (beta subunit) genes (
Vanderwinkel et al., Biochem.Biophys.Res Commun. 33:902-908 (1968);
Korolev et al., Acta Crystallogr.D Biol Crystallogr. 58:2116-2121, (2002)). This enzyme has also been shown to transfer the CoA moiety to acetate, from a
variety of branched and linear acyl-CoA substrates, including isobutyrate (
Matthies et al., Appl Environ Microbiol 58:1435-1439 (1992)), valerate (Vanderwinkel et al.,
supra) and butanoate (Vanderwinkel et al.,
supra), Similar enzymes exist in
Corynebacterium glutamicum ATCC 13032 (
Duncan et al., Appl Environ Microbiol 68:5186-5190 (2002)),
Clostridium acetobutylicum (
Cary et al., Appl Environ Microbiol 56:1576-1583 (1990)), and
Clostridium saccharoperbutylacetionicum (
Kosaka et al., Biosci.Biotechnol Biochem. 71:58-68 (2007)). These proteins are identified below.
Protein |
GenBank ID |
GI Number |
Organism |
atoA |
P76459.1 |
2492994 |
Escherichia coli K12 |
atoD |
P76458.1 |
2492990 |
Escherichia coli K12 |
actA |
YP_226809.1 |
62391407 |
Corynebacterium glutamicum ATCC 13032 |
cg0592 |
YP_224801.1 |
62389399 |
Corynebacterium glutamicum ATCC 13032 |
ctfA |
NP_149326.1 |
15004866 |
Clostridium acetobutylicum |
ctfB |
NP_149327.1 |
15004867 |
Clostridium acetobutylicum |
ctfA |
AAP42564.1 |
31075384 |
Clostridium saccharoperbutylacetonicum |
ctfB |
AAP42565.1 |
31075385 |
Clostridium saccharoperbutylacetonicum |
[0487] Additional exemplary transferase candidates are catalyzed by the gene products of
cat1, cat2, and
cat3 of
Clostridium kluyveri which have been shown to exhibit succinyl-CoA, 4-hydroxybutyryl-CoA, and butyryl-CoA
transferase activity, respectively (Seedorf et al.,
supra; Sohling et al., Eur.J Biochem. 212:121-127 (1993);
Sohling et al., J Bacteriol. 178:871-880 (1996)). Similar CoA transferase activities are also present in
Trichomonas vaginalis (
van Grinsven et al., J.Biol.Chem. 283:1411-1418 (2008)) and
Trypanosoma brucei (
Riviere et al., J.Biol.Chem. 279:45337-45346 (2004)). These proteins are identified below,
Protein |
GenBank ID |
GI Number |
Organism |
cat1 |
P38946.1 |
729048 |
Clostridium kluyveri |
cat2 |
P38942.2 |
172046066 |
Clostridium kluyveri |
cat3 |
EDK35586.1 |
146349050 |
Clostridium kluyveri |
TVAG_395550 |
XP_001330176 |
123975034 |
Trichomonas vaginalis G3 |
Tb11.02.0290 |
XP_828352 |
71754875 |
Trypanosoma brucei |
[0488] The glutaconate-CoA-transferase (EC 2.8.3.12) enzyme from anaerobic bacterium
Acidaminococcus fermentans reacts with diacid glutaconyl-CoA and 3-butenoyl-CoA (
Mack et al., FEBS Lett. 405:209-212 (1997)). The genes encoding this enzyme are
gctA and
gctB. This enzyme has reduced but detectable activity with other CoA derivatives including
glutaryl-CoA, 2-hydroxyglutaryl-CoA, adipyl-CoA and acrylyl-CoA (
Buckel et al., Eur.J.Biochem. 118:315-321 (1981)). The enzyme has been cloned and expressed in
E. coli (
Mack et al., Eur.J.Bioehem. 226:41-51 (1994)). These proteins are identified below.
Protein |
GenBank ID |
GI Number |
Organism |
gctA |
CAA57199.1 |
559392 |
Acidaminococcus fermentans |
gctB |
CAA57200.1 |
559393 |
Acidaminococcus fermentans |
[0489] Enzymes in the 3.1.2 family hydrolyze acyl-CoA molecules to their corresponding acids.
Such a transformation is required by steps F, O, G, T, H, and E of figure 19 and steps
B and H of Figure 20. Several such enzymes have been described in the literature and
represent suitable candidates for these steps.
[0490] For example, the enzyme encoded by
acot12 from
Rattus norvegicus brain (
Robinson et al., Biochem.Biophys.Res.Commun. 71:959-965 (1976)) can react with butyryl-CoA, hexanoyl-CoA and malonyl-CoA. The human dicarboxylic
acid thioesterase, encoded by
acot8, exhibits activity on glutaryl-CoA, adipyl-CoA, suberyl-CoA, sebacyl-CoA, and dodecanedioyl-CoA
(
Westin et al., J.Biol.Chem. 280.38125-38132 (2005)). The closest
E.
coli homolog to this enzyme,
tesB, can also hydrolyze a range of CoA thiolesters (
Naggert et al., J Biol Chem 266:11044-11050 (1991)). A similar enzyme has also been characterized in the rat liver (
Deana R., Biochem Int 26:767-773 (1992)). Additional enzymes with hydrolase activity in
E. coli include
ybgC,
paaI, and
ybdB (
Kuznetsova, et al., FEMS Microbiol Rev, 2005, 29(2):263-279;
Song et al., J Biol Chem, 2006, 281(16):11028-38). Though its sequence has not been reported, the enzyme from the mitochondrion of
the pea leaf has a broad substrate specificity, with demonstrated activity on acetyl-CoA,
propionyl-CoA, butyryl-CoA, palmitoyl-CoA, oleoyl-CoA, succinyl-CoA, and crotonyl-CoA
(
Zeiher et al., Plant.Physiol. 94:20-27 (1990)) The acetyl-CoA hydrolase,
ACH1, from
S. cerevisiae represents another candidate hydrolase (
Buu et al., J. Biol.Chem. 278:17203-17209 (2003)).
Protein |
GenBank Accession # |
GI# |
Organism |
acot12 |
NP_370103.1 |
18543355 |
Rattus norvegicus |
tesB |
NP_414986 |
16128437 |
Escherichia coli |
acot8 |
CAA15502 |
3191970 |
Homo sapiens |
acot8 |
NP_570112 |
51036669 |
Rattus norvegicus |
tesA |
NP_415027 |
16128478 |
Escherichia coli |
ybgC |
NP_415264 |
16128711 |
Escherichia coli |
paaI |
NP_415914 |
16129357 |
Escherichia coli |
ybdB |
NP_415129 |
16128580 |
Escherichia coli |
ACH1 |
NP_009538 |
6319456 |
Saccharomyces cerevisiae |
[0491] Yet another candidate hydrolase is the glutaconate CoA-transferase from
Acidaminococcus fermentans. This enzyme was transformed by site-directed mutagenesis into an acyl-CoA hydrolase
with activity on glutaryl-CoA, acetyl-CoA and 3-butenoyl-CoA (
Mack et al., FEBS.Lett, 405:209-212 (1997)). This suggests that the enzymes encoding succinyl-CoA:3-ketoacid-CoA transferases
and acetoacetyl-CoA:acetyl-CoA transferases may also serve as candidates for this
reaction step but would require certain mutations to change their function.
Protein |
GenBank Accession # |
GI# |
Organism |
gctA |
CAA57199 |
559392 |
Acidaminococcus fermentans |
gctB |
CAA57200 |
559393 |
Acidaminococcus fermentans |
[0492] Additional hydrolase enzymes include 3-hydroxyisobutyryl-CoA hydrolase which has
been described to efficiently catalyze the conversion of 3-hydroxyisobutyryl-CoA to
3-hydroxyisobutyrate during valine degradation (
Shimomura et al., J Biol Chem. 269:14248-14253 (1994)). Genes encoding this enzyme include
hibch of
Rattus norvegicus (
Shimomura et al., Methods Enzymol. 324:229-240 (2000)) and
Homo sapiens (Shimomura et al.,
supra). Similar gene candidates can also be identified by sequence homology, including
hibch of
Saccharomyces cerevisiae and
BC_2292 of
Bacillus cereus.
Protein |
GenBank Acession # |
GI# |
Organism |
hibch |
Q5XIE6.2 |
146324906 |
Rattus norvegicus |
hibch |
Q6NVY1.2 |
146324905 |
Homo sapiens |
hibch |
P28817.2 |
2506374 |
Saccharomyces cerevisiae |
BC_2292 |
AP09256 |
29895975 |
Bacillus cereus |
[0493] Decarboxylase enzymes in the EC class 4.1.1 are required to catalyze steps U,Y,V,
and X of Figure 19, steps D,J,M, and N of figure 20, and steps M,N, and P of figure
21. Candidate decarboxylase enzymes have been described earlier in this application.
[0494] The hydration of a double bond can be catalyzed by hydratase enzymes in the 4.2.1
family of enzymes. The removal of water to form a double bond is catalyzed by dehydratase
enzymes in the 4.2.1 family of enzymes. Hydratase enzymes are sometimes reversible
and also catalyze dehydration. Dehydratase enzymes are sometimes reversible and also
catalyze hydration. The addition or removal of water from a given substrate is required
by steps S, K, L, R, D, C, J, Q, and W in Figure 19, by step F in Figure 20, and by
steps E, F, and O in Figure 21. Several hydratase and dehydratase enzymes have been
described in the literature and represent suitable candidates for these steps.
[0495] For example, many dehydratase enzymes catalyze the alpha, beta-elimination of water
which involves activation of the alpha-hydrogen, by an electron-withdrawing carbonyl,
carboxylate, or CoA-thiol ester group and removal of the hydroxyl group from the beta-position
(
Bucket et al, J Bacteriol, 117:1248-60 (1974);
Martins et al, PNAS 101:15645-9 (2004)), Exemplary enzymes include 2-(hydroxymethyl)glutarate dehydratase (EC 4.2.1.-),
fumarase (EC 4.2.1.2), 3-dehydroquinate dehydratase (EC 4.2.1.10), cyclohexanone hydratase
(EC 4.2.1.-) and 2-keto-4-pentenoate dehydratase (EC 4.2.1.80), citramalate hydrolyase
and dimethylmaleate hydratase.
[0496] 2-(Hydroxymethyl)glutarate dehydratase is a [4Fe-4S]-containing enzyme that dehydrate
2-(hydroxymethyl)glutarate to 2-methylene-glutarate, studied for its role in nicontinate
catabolism in
Eubacterium barkeri (formerly
Clostridium barkeri) (
Alhapel et al., Proc Natl Acad Sci 103:12341-6 (2006)). Similar enzymes with high sequence homology are found in
Bacteroides capillosus, Anaerotruncus colihominis, and
Natranaerobius thermophilius. These enzymes are homologous to the alpha and beta subunits of [4Fe-4S]-containing
bacterial serine dehydratases (e.g.,
E. coli enzymes encoded by
tdcG, sdhB, and
sdaA). An enzyme with similar functionality in
E. barkeri is dimethylmaleate hydratase, a reversible Fe
2+-dependent and oxygen-sensitive enzyme in the aconitase family that hydrates dimethylmaeate
to form (2R,3S)-2,3-dimethylmalate. This enzyme is encoded by
dmdAB (
Alhapel et al., Proc Natl Acad Sci U S A 103:12341-6 (2006);
Kollmann-Koch et al., Hoppe Seylers.Z.Physiol Chem. 365:847-857 (1984)).
Protein |
GenBank ID |
GI Number |
Organism |
hmd |
ABC88407.1 |
86278275 |
Eubacterium barkeri |
BACCAP_02294 |
ZP_02036683.1 |
154498305 |
Bacteroides capillosus |
ANACOL_02527 |
ZP_02443222.1 |
167771169 |
Anaerotruncus colihominis |
NtherDRAFT_2368 |
ZP_02852366.1 |
169192667 |
Natranaerobius thermophilus |
dmdA |
ABC88408 |
86278276 |
Eubacterium barkeri |
dmdB |
ABC88409 |
86278277 |
Eubacterium barkeri |
[0497] Fumarate hydratase (EC 4.2.1.2) enzymes naturally catalyze the reversible hydration
of fumarate to malate. Although the ability of fumarate hydratase to react with 3-oxobtitanol
as a substrate has not been described in the literature, a wealth of structural information
is available for this enzyme and other researchers have successfully engineered the
enzyme to alter activity, inhibition and localization (Weaver, 61:1395-1401 (2005)).
E. coli has three fumarases: FumA, FumB, and FumC that are regulated by growth conditions.
FumB is oxygen sensitive and only active under anaerobic conditions. FumA is active
under microanaerobic conditions, and FumC is the only active enzyme in aerobic growth
(
Tseng et al., J Bacteriol, 183:461-467 (2001); Woods et al., 954:14-26 (1988);
Guest et al., J Gen Microbiol 131:2971-2984 (1985)). Additional enzyme candidates are found in
Campylobacter jejuni (
Smith et al., Int.J Biochem.Cell Biol 31:961-975 (1999)),
Thermus thermophilus (
Mizobata et al, Arch.Biochem.Biophys. 355:49-55 (1998)) and
Rattus norvegicus (
Kobayashi et al., J. Biochem, 89:923-1931 (1981)). Similar enzymes with high sequence homology include
fum1 from
Arabidopsis thaliana and
fumC from
Corynebacterium glutamicum. The
MmcBC fumarase from
Pelotomaculum thermopropionicum is another class of fumarase with two subunits (
Shimoyama et al., FEMS Microbiol Lett, 270:207-213 (2007)).
Protein |
GenBank ID |
GI Number |
Organism |
fumA |
NP_416129.1 |
16129570 |
Escherichia coli |
fumB |
NP_418546.1 |
16131948 |
Esceherichia coli |
fumC, |
NP_416128.1 |
16129569 |
Escherichia coli |
fumC |
069294 |
9789756 |
Campylobacter jejunini |
fumC |
P84127 |
75427690 |
Thermus thermophilus |
fumH |
P14408 |
1206115 |
Rattus norvegicus |
fumI |
P93033 |
39931311 |
Arabidopsis thaliana |
fumC |
Q8NRN8 |
39931596 |
Corynebacterium glutamicum |
MmcB |
YP_001211906 |
147677691 |
Pelotomaculum thermopropionicum |
MmcC |
YP_001211907 |
147677692 |
Pelotomaculum thermopropionicum |
[0499] (
Wang et al., FEBS J 272:966-974 (2005)). A closely related enzyme, 2-oxohepta-4-ene-1,7-dioate hydratase, participates
in 4-hydroxyphenylacetic acid degradation, where it converts 2-oxo-hept-4-ene-1,7-dioate
(OHED) to 2-oxo-4-hydroxy-hepta-1,7-dioate using magnesium as a cofactor (
Burks et al., J.Am.Chem.Soc. 120: (1999)). OHED hydratase enzyme candidates have been identified and characterized in
E. coli C (
Roper et al., Gene 156:47-51 (1995);
Izumi et al., J Mol.Biol, 370:899-911 (2007)) and
E. coli W (
Prieto et al., J Bacteriol. 178:111-120 (1996)). Sequence comparison reveals homologs in a wide range of bacteria, plants and animals.
Enzymes with highly similar sequences are contained in
Klebsiella pneumonia (91% identity, eval = 2e-138) and
Salmonella enterica (91% identity, eval = 4e-138), among others.
Protein |
GenBank Accession No. |
GI No. |
Organism |
mhpD |
AAC73453.2 |
87081722 |
Escherichia coli |
cmtF |
AAB62293.1 |
1263188 |
Pseudomonas putida |
todG |
AAA61942.1 |
485738 |
Pseudomonas putida |
cnbE |
YP_001967714.1 |
190572008 |
Comamonas sp. CNB-1 |
mhpD |
Q13VU0 |
123358582 |
Burkholderia xenovorans |
hpcG |
CAA57202.1 |
556840 |
Escherichia coli C |
hpaH |
CAA86044.1 |
757830 |
Escherichia coli W |
hpaH |
ABR80130.1 |
150958100 |
Klebsiella pneumoniae |
Sari_01896 |
ABX21779.1 |
160865156 |
Salmonella enterica |
[0500] Another enzyme candidate is citramalate hydrolyase (EC 4.2.1.34), an enzyme that
naturally dehydrates 2-methylmalate to mesaconate. This enzyme has been studied in
Methanocaldococcus jannaschii in the context of the pyruvate pathway to 2-oxobutanoate, where it has been shown
to have a broad substrate specificity (
Drevland et al., J Bacteriol. 189:4391-4400 (2007)). This enzyme activity was also detected in
Clostridium tetanomorphum, Morganella morganii, Citrobacter amalonaticus where it is thought to participate in glutamate degradation (
Kato et al., Arch.Microbiol 168:457-463 (1997)). The
M. jannaschii protein sequence does not bear significant homology to genes in these organisms.
Protein |
GenBank ID |
GI Number |
Organism |
leuD |
Q58673.1 |
3122345 |
Methanocaldococcus jannaschii |
[0501] Dimethylmateate hydratase (EC 4.2.1.85) is a reversible Fe
2+-dependent and oxygen-sensitive enzyme in the aconitase family that hydrates dimethylmaeate
to form (2R,3S)-2,3-dimethylmalate. This enzyme is encoded by
dmdAB in
Eubacterium barkeri (Alhapel et al.,
supra; Kollmann-Koch et al., Hoppe Scylers.ZPhysiol Chem. 365:847-857 (1984)).
Protein |
GenBank ID |
GI Number |
Organism |
dmdA |
ABC88408 |
86278276 |
Eubactrium backeri |
dmdB |
ABC88409.1 |
86278277 |
Eubacterium barkeri |
[0502] Oleate hydratases represent additional suitable candidates as suggested in
WO2011076691. These are particularly useful for step W of Figure 19 and step O of Figure 2 1 .
Examples include the following proteins.
Protein |
GenBank ID |
GI Number |
Organism |
OhyA |
ACT54545.1 |
254031735 |
Elizabethkingia meningoseptica |
HMPREF0841_1446 |
ZP_07461147.1 |
306827879 |
Streptococcus pyogenes, ATCC 10782 |
P700755_13397 |
ZP_01252267.1 |
9.1215295 |
Psychroflexus torquis ATCC 700755 |
RPB_2430 |
YP_486046.1 |
86749550 |
Rhodopseudomonas palustris |
[0503] Enoyl-CoA hydratases (EC 4.2.1.17) catalyze the dehydration of a range of 3-hydroxyacyl-CoA
substrates (
Roberts et al., Arch.Microbiol 17:99-108 (1978);
Agnihotri et al., Bioorg.Med.Chem. 11:9-20 (2003);
Conrad et al., J Bacteriol, 118:103-111 (1974)). The enoyl-CoA hydratase of
Pseudomonas putida, encoded by ech, catalyzes the conversion of 3-hydroxybutyryl-CoA to crotonyl-CoA
(
Roberts et al. Arch.Microbiol 117:99-108 (1978)). This transfonnation is also catalyzed by the
crt gene product of
Clostridium acetobutylicum, the
crt1 gene product of
C. kluyveri, and other clostridial organisms
Atsumi et al., Metab Eng 10:305-3,11 (2008);
Boynton ot al., J Bacteriol. 178:3015-3024 (1996);
Hilimer et al., FEBS Lett. 21:351-354 (1972)). Additional enoyl-CoA hydratase candidates are
phaA and
phaB, of
P. puttida, and
paaA and
paaB from
P. fluorescens (
Olivera et al., Proc.Natl.Acad.Sci U.S.A 95:6419-6424 (1998)). The gene product of
pimF in
Rhodopseudomonas palustris is predicted to encode an enoyl-CoA hydratase that participates in pimeloyl-CoA degradation
(
Harrison et al,, Microbiology 151:727-736 (2005)). Lastly, a number of
Escherichia coli genes have been shown to demonstrate enoyl-CoA hydratase functionality including
maoC (
Park et al., J Bacteriol. 185:5391-5397 (2003)),
paaF (
Ismail et al., Eur.J Biochem. 270:3047-3054 (2003);
Park et al., App/.Biochem.Biotechnol 113 -116:33 5-346 (2004);
Park et al., Biotechnol Bioeng 86:681-686 (2004)) and
paaG (
Ismail et al., Eur.J Bichem. 270:3047-3054 (2003);
Park and Lee, Appl.Biochem.Biotechnol 113-116:335-346 (2004);
Park and Yup, Biotechnol Bioeng 86:681-686 (2004)).
Protein |
CenBank Accession No. |
GI No. |
Organism |
ech |
NP_745498.1 |
26990073 |
Pseudomonas putida |
crt |
NP_349318.1 |
15895969 |
Clostridium acetobutylicum |
crtI |
YP_001393856 |
153953091 |
Clostridium |
phaA |
ABF82233.1 |
26990002 |
Pseudomonas putida |
phaB |
ABF82234.1 |
26990001 |
Pseudomonas putida |
paaA |
NP_745427.1 |
106636093 |
Pseudomonas fluorescens |
paaB |
NP_745426.1 |
106636094 |
Pseudomonas fluorescens |
maoC |
NP_415905.1 |
16129348 |
Escherichia coli |
paaF |
NP_415911.1 |
16129354 |
Escherichia coli |
paaF |
NP_415912.1 |
16129355 |
Escherichia coli |
[0505] The conversion of acyl-CoA substrates to their acid products can be catalyzed by
a CoA acid-thiol ligase or CoA synthetase in the 6.2.1 family of enzymes, several
of which are reversible. Several reactions shown in Figures 19-20 are catalyzed by
acid-thiol ligase enzymes. These reactions include Steps F, O, G, T, H, and E of Figure
19 and Steps B and H of Figure 20. Several enzymes catalyzing CoA acid-thiol ligase
or CoA synthetase activities have been described in the literature and represent suitable
candidates for these steps.
[0506] For example, ADP-forming acetyl-CoA synthetase (ACD, EC 6.2.1.13) is an enzyme that
couples the conversion of acyl-CoA esters to their corresponding acids with the concomitant
synthesis of ATP. ACD I from
Archaeoglobus fulgidus, encoded by AF1211, was shown to operate on a variety of linear and branched-chain
substrates including isobutyrate, isopentanoate, and fumarate (
Musfeldt et al., J Bacteriol. 184:636-644 (2002)). A second reversible ACD in
Archaeoglobus fulgidus, encoded by AF1983, was also shown to have a broad substrate range with high activity
on cyclic compounds phenylacetate and indoleacetate (
Musfeldt and Schonheit, J Bacteriol. 184:636-644 (2002)). The enzyme from
Haloarcula marismortui (annotated as a succinyl-CoA synthetase) accepts propionate, butyrate, and branched-chain
acids (isovalerate and isobutyrate) as substrates, and was shown to operate in the
forward and reverse directions (
Brasen et al., Arch Microbiol 182:277-287 (2004)). The ACD encoded by
PAE3250 from hyperthermophilic crenarchaeon
Pyrobaculum, aerophilum showed the broadest substrate range of all characterized ACDS, reacting with acetyl-CoA,
isobutyryl-CoA (preferred substrate) and phenylacetyl-CoA (Brasen et al,
supra). Directed evolution or engineering can be used to modify this enzyme to operate at
the physiological temperature of the host organism. The enzymes from
A. fylgidus,
H. marismortui and P. aerophilum have all been cloned, functionally expressed, and characterized in
E.
coli (Brasen and Schonheit,
supra; Musfeldt and Schonheit, J Bacteriol. 184:636-644 (2002)). An additional is succinyl-CoA synthetase, encoded by
sucCD of
E.
coli and
LSC1 and
LSC2 genes of
Saccharomyces cerevisiae. These enzymes catalyze the formation of succinyl-CoA from succinate with the concomitant
consumption of one ATP in a reaction which is reversible
in vivo (
Buck et al., Biochemistry 24:6245-6252 (1985)). The acyl CoA ligase from
Pseudomonas putida has been demonstrated to work on several aliphatic substrates including acetic, propionic,
butyric, valeric, hexanoic, heptanoic, and octanoic acids and on aromatic compounds
such as phenylacetic and phenoxyacetic acids (
Fernandez-Valverde et al., Appl.Environ.Microbiol. 59:1149-1154 (1993)). A related enzyme, malonyl CoA synthetase (6.3.4.9) from
Rhizobium leguminosarum could convert several diacids, namely, ethyl-, propyl-, allyl-, isopropyl-, dimethyl-,
cyclopropyl-, cyclopropylmethylene-, cyclobutyl-, and benzyl-malonate into their corresponding
monothioesters (
Pohl et al., J.Am.Chem.Soc. 123:5822-5823 (2001)).
Protein |
GenBank ID |
GI Number |
Organism |
AF1211 |
NP_070039.1 |
11498810 |
Archaeoglobus fulgidus |
AF1983 |
NP_070807.1 |
11499565 |
Archaeoglobus fulgidus |
Scs |
YP_135572.1 |
55377722 |
Haloarcula marismortui |
PAE3250 |
NP_560604.1 |
18313937 |
Pyrobaculum aerophilum str. IM2 |
sucC |
NP_415256.1 |
16128703 |
Escherichia coli |
sucD |
AAC73823.1 |
1786949 |
Escherichia coli |
LSC1 |
NP_01485 |
6324716 |
Saccharomyces cerevisiae |
LSC2 |
NP_11760 |
6321683 |
Saccharomyces cerevisiae |
paaF |
AAC24333.2 |
22711873 |
Pseudomonas putida |
matB |
AAC83455.1 |
3982573 |
Rhizobium leguminosarum |
[0507] Another candidate enzyme for these steps is 6-carboxyhexanoate-CoA ligase, also known
as pimeloyl-CoA ligase (EC 6.2.1.14), which naturally activates pimelate to pimeloyl-CoA
during biotin biosynthesis in gram-positive bacteria. The enzyme from
Pseudomonas mendocina, closed into
E. coli, was shown to accept the alternate substrates hexanedioate and nonanedioate (
Binieda et al., Biochem.J 340 (Pt 3):793-801 (1999)). Other candidates are found in
Bacillus subtilis (
Bower et al., J Bacteriol. 178:4122-4130 (1996)) and
Lysinibacillus sphaericus (formerly
Bacillus sphaericus (
Ploux et al., Biochem.J 287 (Pt 3):685-690 (1992)).
Protein |
GenBank ID |
GI Number |
Organism |
bioW |
NP_390902.2 |
50812281 |
Bacillus subtilis |
bioW |
CAA10043.1 |
3850837 |
Pseudomonas mendocina |
bioW |
P22822.1 |
115012 |
Bacillus sphaericus |
[0509] Like enzymes in other classes, certain enzymes in the EC class 6.2.1 have been determined
to have broad substrate specificity. The acyl CoA ligase from
Pseudomonas putida has been demonstrated to work on several aliphatic substrates including acetic, propionic,
butyric, valeric, hexanoic, heptanoic, and octanoic acids and on aromatic compounds
such as phenylacetic and phenoxyacetic acids (
Fernandez-Valverde et al., Applied and Environmental Microbiology 59:1149-1154 (1993)). A rotated enzyme, malonyl CoA synthetase (6.3.4.9) from
Rhizobium, trifolii could convert several diacids, namely, ethyl-, propyl-, allyl-, isopropyl-, dimethyl-,
cyclopropyl-, cyclopropylmethylene-, cyclobutyl-, and benzyl-malonate into their corresponding
monothioesters (
Pohl et al., J.Am.Chem.Soc. 123:5822-5823 (2001)).
[0510] Throughout this application various publications have been referenced. The disclosures
of these publications in their entireties, including GenBank and CI number publications,
are hereby incorporated by reference in this application in order to more fully describe
the state of the art to which this invention pertains. Although the invention has
been described with reference to the examples provided above, it should be understood
that various modifications can be made without departing from the spirit of the invention.
[0511] Preferred Items:
- 1. A non-naturally occurring microbial organism, comprising a microbial organism having
a toluene pathway comprising at least one exogenous nucleic acid encoding a toluene
pathway enzyme expressed in a sufficient amount to produce toluene, said toluene,
pathway selected from (A) 1) one or both of phenylalanine aminotransferase and phenylalanine
oxidoreductase (deaminating), 2) phenylpyruvate decarboxylase, and 3) phenylacetaldehyde
decarbonylase; (B) 1) one or more of phenylalanine aminotransferase and phenylalanine
oxidoreductase (deaminating), 2) phenylpyruvate decarboxylase, 3) one or more of phenylacetaldehyde
dehydrogenase and phenylacetaldehyde oxidase, and 4) phenylacetate decarboxylase;
(C) 1) one or more of phenylalanine aminotransferase and phenylalanine oxidoreductase
(deaminating), 2) phenylpyruvate oxidase, and 3) phenylacetate decarboxylase; and
(D) 1) phenylalanine aminotransferase and/or phenylalanine oxidoreductase (deaminating),
2) phenylpyruvate oxidase and 3) phenylacetate decarboxylase.
- 2. The non-naturally occurring microbial organism of item 1, wherein said microbial
organism comprises two exogenous nucleic acids each encoding a toluene, pathway enzyme.
- 3. The non-naturally occurring microbial organism of item 1, wherein said microbial
organism comprises three exogenous nucleic acids each encoding a toluene pathway enzyme.
- 4. The non-naturally occurring microbial organism of item 3, wherein said three exogenous
nucleic acids encode 1) phenylalanine aminotransferase or phenylalanine oxidoreductase
(deaminating), 2) phenylpyruvate decarboxylase, 3) phenylacetaldehyde decarbonylase.
- 5. The non-naturally occurring microbial organism, of item 1, wherein said microbial
organism comprises four exogenous nucleic acids each encoding a toluene pathway enzyme.
- 6. The non-naturally occurring microbial organism of item 5, wherein said four exogenous
nucleic acids encode 1) phenylalanine aminotransferase or phenylalanine oxidoreductase
(deaminating), 2) phenylpyruvate decarboxylase, 3) phenylacetaldehyde dehydrogenase
or oxidase, and 4) phenylacetate decarboxylase.
- 7. The non-naturally occurring microbial organism of item 1, wherein said at least
one exogenous nucleic acid is a heterologous nucleic acid.
- 8. The non-naturally occurring microbial organism of item 1, wherein said non-naturally
occurring microbial organism is in a substantially anaerobic culture medium.
- 9. A method for producing toluene, comprising culturing a non-naturally occurring
microbial organism having a toluene pathway, said pathway comprising at least one
exogenous nucleic acid encoding a toluene pathway enzyme expressed in a sufficient
amount to produce toluene, under conditions and for a sufficient period of time to
produce toluene, said toluene pathway selected from (A) 1) one or both of phenylalanine
aminotransferase and phenylalanine oxidoreductase (deaminating), 2) phenylpyruvate
decarboxylase, and 3) phenylacetaldehyde decarbonylase; (B) 1) one or more of phenylalanine
aminotransferase and phenylalanine oxidoreductase (deaminating), 2) phenylpyruvate
decarboxylase, 3) one or more of phenylacetaldehyde dehydrogenase and phenylacetaldehyde
oxidase, and 4) phenylacetate decarboxylase; (C) one or more of phenylalanine aminotransferase
and phenylalanine oxidoreductase (deaminating), 2) phenylpyruvate oxidase, and 3)
phenylacetate decarboxylase; and (D) 1) phenylalanine aminotransferase and/or phenylalanine
oxidoreductase (deaminating), 2) phenylpyruvate oxidase and 3) phenylacetate decarboxylase.
- 10. The method of item 9, wherein said non-naturally occurring microbial organism
is in a substantially anaerobic culture medium.
- 11. The method of item 9, wherein said microbial organism comprises two exogenous
nucleic acids each encoding a toluene pathway enzyme.
- 12. The method of item 9, wherein said microbial organism comprises three exogenous
nucleic acids each encoding a toluene pathway enzyme.
- 13. The method of item 12, wherein said three exogenous nucleic acids encoded 1) phenylalanine
aminotransferase or phenylalanine oxidoreductase (deaminating), 2) phenylpyruvate
decarboxylase, and 3) phenylacetaldehyde decarbonylase.
- 14. The method of item 9, wherein said microbial organism comprises four exogenous
nucleic acids each encoding a toluene pathway enzyme.
- 15. The method of item 14, wherein said four exogenous nucleic acids encode 1) phenylalanine
aminotransferase or phenylalanine oxidoreductase (deaminating), 2) phenylpyruvate
decarboxylase, 3) phenylacetaldehyde dehydrogenase or oxidase, and 4) phenylacetate
decarboxylase.
- 16. The method of item 9, wherein said at least one exogenous nucleic acid is a heterologous
nucleic acid.
- 17. A non-naturally occurring microbial organism, comprising a microbial organism
having a benzene pathway comprising at least one exogenous nucleic acid encoding a
benzene pathway enzyme expressed in a sufficient amount to produce benzene, said benzene
pathway comprising a phenylalanine benzene-lyase.
- 18. The non-naturally occurring microbial organism of item 17, wherein said at least
one exogenous nucleic acid is said phenylalanine benzene-lyase.
- 19. The non-naturally occurring microbial organism of item 17, wherein said at least
one exogenous nucleic acid is a heterologous nucleic acid.
- 20. The non-naturally occurring microbial organism of item 17, wherein said non-naturally
occurring microbial organism is in a substantially anaerobic culture medium.
- 21. A method for producing benzene, comprising culturing a non-naturally occurring
microbial organism having a benzene pathway, said pathway comprising at least one
exogenous nucleic acid encoding a benzene pathway enzyme expressed in a sufficient
amount to produce benzene, under conditions and for a sufficient period of time to
produce benzene, said benzene pathway comprising a phenylalanine benzene-lyase.
- 22. The method of item 21, wherein said at least one exogenous nucleic acid is said
phenylalanine benzene-lyase.
- 23. The method of item 21, wherein said at least one exogenous nucleic acid is a heterologous
nucleic acid.
- 24. The method of item 21, wherein said non-naturally occurring microbial organism
is in a substantially anaerobic culture medium.
- 25. A non-naturally occurring microbial organism, comprising a microbial organism
having a styrene pathway comprising at least one exogenous nucleic acid encoding a
styrene pathway enzyme expressed in a sufficient amount to produce styrene, said styrene
pathway selected from (A) 1) benzoyl-CoA acetyltransferase, 2) one or more of 3-oxo-3-phenylpropionyl-CoA
synthetase, transferase, and hydrolase, 3) benzoyl-acetate decarboxylase, 4) acetopheone
reductase, and 5) 1-phenylethanol dehydratase; or (B) 1) benzoyl-CoA acetyltransferase,
2) phosphotrans-3-oxo-3-phenylpropionylase, 3) benzoyl-acetate kinase, 4) benzoyl-acetate
decarboxylase, 5) acetopheone reductase, and 6) 1-phenylethanol dehydratase.
- 26. The non-naturally occurring microbial organism of item 25, wherein said microbial
organism comprises two exogenous nucleic acids each encoding a styrene pathway enzyme.
- 27. The non-naturally occurring microbial organism of item 25, wherein said microbial
organism comprises three exogenous nucleic acids each encoding a styrene pathway enzyme.
- 28. The non-naturally occurring microbial organism of item 25, wherein said microbial
organism comprises four exogenous nucleic acids each encoding a styrene pathway enzyme.
- 29. The non-naturally occurring microbial organism of item 25, wherein said microbial
organism comprises five exogenous nucleic acids each encoding a styrene pathway enzyme.
- 30. The non-naturally occurring microbial organism of item 29, wherein said five exogenous
nucleic acids encode 1) benzoyl-CoA acetyltransferase, 2) one of 3-oxo-3-phenylpropionyl-CoA
synthetase, transferase, and hydrolase, 3) benzoyl-acetate decarboxylase, 4) acetopheone
reductase, and 5) 1-phenylethanol dehydratase.
- 31. The non-naturally occurring microbial organism of item 25, wherein said microbial
organism comprises six exogenous nucleic acids each encoding a styrene pathway enzyme.
- 32. The non-naturally occurring microbial organism of item 31, wherein said six exogenous
nucleic acids encode 1) benzoyl-CoA acetyltransferase, 2) phosphotrans-3-oxo-3-phenylpropionylase,
3) benzoyl-acetate kinase, 4) benzoyl-acetate decarboxylase, 5) acetopheone reductase,
and 6) 1-phenylethanol dehydratase.
- 33. The non-naturally occurring microbial organism of item 25, wherein said at least
one exogenous nucleic acid is a heterologous nucleic acid.
- 34. The non-naturally occurring microbial organism of item 25, wherein said non-naturally
occurring microbial organism is in a substantially anaerobic culture medium.
- 35. A method for producing styrene, comprising culturing a non-naturally occurring
microbial organism having a styrene pathway, said pathway comprising at least one
exogenous nucleic acid encoding a styrene pathway enzyme expressed in a sufficient
amount to produce styrene, under conditions and for a sufficient period of time to
produce styrene, said styrene pathway selected from (A) 1) benzoyl-CoA acetyltransferase,
2) one or more of 3-oxo-3-phenylpropionyl-CoA synthetase, transferase, and hydrolase,
3) benzoyl-acetate decarboxylase, 4) acetopheone reductase, and 5) 1-phenylethanol
dehydratase; or (B) 1) benzoyl-CoA acetyltransferase, 2) phosphotrans-3-oxo-3-phenylpropionylase,
3) benzoyl-acetate kinase, 4) benzoyl-acetate decarboxylase, 5) acetopheone reductase,
and 6) 1-phenylethanol dehydratase.
- 36. The method of item 35, wherein said non-naturally occurring microbial organism
is in a substantially anaerobic culture medium.
- 37. The method of item 35, wherein said microbial organism comprises two exogenous
nucleic acids each encoding a styrene pathway enzyme.
- 38. The method of item 35, wherein said microbial organism comprises three exogenous
nucleic acids each encoding a styrene pathway enzyme.
- 39. The method of item 35, wherein said microbial organism comprises four exogenous
nucleic acids each encoding a styrene pathway enzyme.
- 40. The method of item 35, wherein said microbial organism comprises five exogenous
nucleic acids each encoding a styrene pathway enzyme.
- 41. The method of item 40, wherein said five exogenous nucleic acids encode 1) benzoyl-CoA
acetyltransferase, 2) one of 3-oxo-3-phenylpropionyl-CoA synthetase, transferase,
and hydrolase, 3) benzoyl-acetate decarboxylase, 4) acetopheone reductase, and 5)
1-phenylethanol dehydratase.
- 42. The method of item 35, wherein said microbial organism comprises six exogenous
nucleic acids each encoding a styrene pathway enzyme.
- 43. The method of item 42, wherein said six exogenous nucleic acids encode 1) benzoyl-CoA
acetyltransferase, 2) phosphotrans-3-oxo-3-phenylpropionylase, 3) benzoyl-acetate
kinase, 4) benzoyl-acetate decarboxylase, 5) acetopheone reductase, and 6) 1-phenylethanol
dehydratase.
- 44. The method of item 35, wherein said at least one exogenous nucleic acid is a heterologous
nucleic acid.
- 45. A non-naturally occurring microbial organism, comprising a microbial organism
having a 1,3-butadiene pathway comprising at least one exogenous nucleic acid encoding
a 1,3-butadiene pathway enzyme expressed in a sufficient amount to produce 1,3-butadiene,
said 1,3-butadiene pathway selected from (A) 1) trans, trans-muconate decarboxylase and 2) trans-2,4-pentadienoate decarboxylase; (B) 1) cis, trans-muconate civ-decarboxylase and 2) trans-2,4-pentadienoate decarboxylase; (C) 1) cis, trans-muconate trans-decarboxylase 2) cis-2,4-pentadienoate decarboxylase; (D) 1) cis, cis-muconate decarboxylase and 2) cis-2,4-pentadienoate decarboxylase; (E) cis-2,4-pentadienoate decarboxylase; and (F) trans-2,4-pentadienoate decarboxylase.
- 46. The non-naturally occurring microbial organism of item 45, wherein said microbial
organism comprises two exogenous nucleic acids each encoding a 1,3-butadiene pathway
enzyme.
- 47. The non-naturally occurring microbial organism of item 46, wherein said two exogenous
nucleic acids encode a set selected from (A) 1) trans, trans-muconate decarboxylase and 2) trans-2,4-pentadienoate decarboxylase; (B) 1) cis, trans-muconate cis-decarboxylase and 2) trans-2,4-pentadienoate decarboxylase, (C) 1) cis, trans-muconate trans-decarboxylase 2) cis-2,4-pentadienoate decarboxylase; and (D) 1) cis, cis-muconate decarboxylase and 2) cis-2,4-pentadienoate decarboxylase.
- 48. The non-naturally occurring microbial organism of item 45, wherein said at least
one exogenous nucleic acid is a heterologous nucleic acid.
- 49. The non-naturally occurring microbial organism of item 45, wherein said non-naturally
occurring microbial organism is in a substantially anaerobic culture, medium.
- 50. A method for producing 1,3-butadiene, comprising culturing a non-naturally occurring
microbial organism having a 1,3-butadiene pathway, said pathway comprising at least
one exogenous nucleic acid encoding a 1,3-butadiene pathway enzyme expressed in a
sufficient amount to produce 1,3-butadiene, under conditions and for a sufficient
period of time to produce 1,3-butadiene, said 1,3-butadiene pathway selected from
(A) 1) trans, trans-muconate decarboxylase and 2) trans-2,4-pentadienoate decarboxylase; (B) 1) cis, trans-muconate cis-decarboxylase and 2) trans-2,4-pentadienoate decarboxylase; (C) 1) cis, trans-muconate trans-decarboxylase 2) cis-2,4-pentadienoate decarboxylase; (D) 1) cis, cis-muconate decarboxylase and 2) cis-2,4-pentadienoate decarboxylase; (E) cis-2,4-pentadienoate decarboxylase; and (F) trans-2,4-pentadienoate decarboxylase.
- 51. The method of item 50, wherein said non-naturally occurring microbial organism
is in a substantially anaerobic culture medium.
- 52. The method of item 50, wherein said microbial organism comprises two exogenous
nucleic acids each encoding a 1,3-butadiene pathway enzyme.
- 53. The method of item 52, wherein said two exogenous nucleic acids encode a set selected
from (A) 1) trans, trans-muconate decarboxylase and 2) trans-2,4-pentadienoate decarboxylase; (B) 1) cis, trans-muconate cis-decarboxylase and 2) trans-2,4-pentadienoate decarboxylase; (C) 1) cis, trans-muconate trans-decarboxylase 2) cis-2,4-pentadienoate decarboxylase; and (D) 1) cis, cis-muconate decarboxylase and 2) cis-2,4-pentadienoate decarboxylase.
- 54. The method of item 50, wherein said at least one exogenous nucleic acid is a heterologous
nucleic acid.
- 55. A non-naturally occurring microbial organism, comprising a microbial organism
having a (2-hydroxy-4-oxobutoxy)phosphonate pathway comprising at least one exogenous
nucleic acid encoding a (2-hydroxy-4-oxobutoxy)phosphonate pathway enzyme expressed
in a sufficient amount to produce (2-hydroxy-4-oxobutoxy)phosphonate, said (2-hydroxy-4-oxobutoxy)phosphonate
pathway comprising erythrose-4-phosphate dehydratase and (2,4-dioxobutoxy)phosphonate
reductase.
- 56. The non-naturally occurring microbial organism of item 55, wherein said microbial
organism comprises two exogenous nucleic acids each encoding a (2-hydroxy-4-oxobutoxy)phosphonate
pathway enzyme.
- 57. The non-naturally occurring microbial organism of item 56, wherein said two exogenous
nucleic acids encode erythrose-4-phosphate dehydratase and (2,4-dioxobutoxy)phosphonate
reductase.
- 58. The non-naturally occurring microbial organism of item 55, wherein at least one
exogenous nucleic acid is a heterologous nucleic acid.
- 59. The non-naturally occurring microbial organism of item 55, wherein said microbial
organism is cultured anaerobically.
- 60. A non-naturally occurring microbial organism, comprising a microbial organism
having a benzoate pathway comprising at least one exogenous nucleic acid encoding
a benzoate pathway enzyme expressed in a sufficient amount to produce benzoate, said
benzoate pathway comprising 2-dehydro-3-deoxyphosphoheptonate synthase; 3-dehydroquinate
synthase; 3-dehydroquinate dehydratase; shikimate dehydrogenase; shikimate kinase;
3-phosphoshikimate-2-carboxyvinyltransferase; chorismate synthase; and chorismate
lyase.
- 61. The non-natarally occurring microbial organism of item 60, wherein said microbial
organism comprises two exogenous nucleic acids each encoding a benzoate pathway enzyme.
- 62. The non-naturally occurring microbial organism of item 60, wherein said microbial
organism comprises three exogenous nucleic acids each encoding a benzoate pathway
enzyme.
- 63. The non-naturally occurring microbial organism of item 60, wherein said microbial
organism comprises four exogenous nucleic acids each encoding a benzoate pathway enzyme.
- 64. The non-naturally occurring microbial organism of item 60, wherein said microbial
organism comprises five exogenous nucleic acids each encoding a benzoate pathway enzyme.
- 65. The non-naturally occurring microbial organism of item 60, wherein said microbial
organism comprises six exogenous nucleic acids each encoding a benzoate pathway enzyme.
- 66. The non-naturally occurring microbial organism of item 60, wherein Said microbial
organism comprises seven exogenous nucleic acids each encoding a benzoate pathway
enzyme.
- 67. The non-naturally occurring microbial organism of item 60, wherein said microbial
organism comprises eight exogenous nucleic acids each encoding a benzoate pathway
enzyme.
- 68. The non-naturally occurring microbial organism of item 67, wherein said eight
exogenous nucleic acids encode 2-dehydro-3-deoxyphosphoheptonate synthase; 3-dehydroquinate
synthase; 3-dehydroquinate dehydratase; shikimate dehydrogenase; shikimate kinase;
3-phosphoshikimate-2-carboxyvinyltransferase; chorismate synthase; and chorismate
lyase.
- 69. The non-naturally occurring microbial organism of item 60 further comprising a
(2-hydroxy-4-oxobutoxy)phosphonate pathway comprising erythrose-4-phosphate dehydratase
and (2,4-dioxobutoxy)phosphonate reductase.
- 70. The non-naturally occurring microbial organism of item 60, wherein at least one
exogenous nucleic acid is a heterologous nucleic acid.
- 71. The non-naturally occurring microbial organism of item 60, wherein said non-naturally
occurring microbial organism is in a substantially anaerobic culture medium.
- 72. A method for producing benzoate, comprising culturing the non-naturally occurring
microbial organism of item 60 under conditions and for a sufficient period of time
to produce benzoate.
- 73. The method of item 72, wherein said non-naturally occurring microbial organism
is in a substantially anaerobic culture medium.
- 74. The method of item 72, wherein said microbial organism comprises eight exogenous
nucleic acids each encoding a benzoate pathway enzyme.
- 75. The method of item 74, wherein said eight exogenous nucleic acids encode 2-dehydro-3-deoxyphosphoheptonate
synthase; 3-dehydroquinate synthase; 3-dehydroquinate dehydratase; shikimate dehydrogenase;
shikimate kinase; 3-phosphoshikimate-2-carboxyvinyltransferase; chorismate synthase;
and chorismate lyase.
- 76. The method of item 72, wherein said microbial organism further comprises a (2-hydroxy-4-oxobutoxy)phosphonate
pathway comprising erythrose-4-phosphate dehydratase and (2,4-dioxobutoxy)phosphonate
reductase.
- 77. The method of item 72, wherein said at least one exogenous nucleic acid is a heterologous
nucleic acid.
- 78. A non-naturally occurring microbial organism, comprising a microbial organism
having a benzene pathway comprising at least one exogenous nucleic acid encoding a
benzene pathway enzyme expressed in a sufficient amount to produce benzene, said benzene
pathway is selected from a set of pathway enzymes selected from: a) benzoate decarboxylase;
b) benzoate and benzaldehyde decarbonylase; c) benzoate kinase, (benzoyloxy)phosphonate
reductase, and benzaldehyde decarbonylase; d) (benzoyl-CoA synthetase, transferase
and/or hydrolase), phosphotransbenzoylase, (benzoyloxy)phosphonate reductase, and
benzaldehyde decarbonylase; and e) (benzoyl-CoA synthetase, transferase and/or hydrolase),
benzoyl-CoA reductase and benzaldehyde decarbonylase, f) (benzoyl-CoA synthetase,
transferase and/or hydrolase), benzoate decarboxylase, g) benzoyl-CoA reductase and
benzaldehyde decarbonylase, h) phosphotransbenzoylase, (benzoyloxy)phosphonate reductase,
and benzaldehyde decarbonylase, i) phosphotransbenzoylase, benzoate kinase, benzoate
decarboxytase, j) phosphotransbenzoytase, benzoate kinase, benzoate reductase, benzaldehyde
decarbonylase; k) phosphotransbenzoylase, (benzoytoxy)phosphonate reductase, and benzaldehyde
decarbonytase; and 1) benzoyl-CoA reductase and benzaldehyde decarbonylase.
- 79. The non-naturally occurring microbial organism of item 78, wherein said benzene
pathway comprises benzoate decarboxylase.
- 80. The non-naturally occurring microbial organism of item 78, wherein said benzene
pathway comprises benzoate reductase and benzaldehyde decarbonylase
- 81. The non-naturally occurring microbial organism of item 78, wherein said benzene
pathway comprises benzoate kinase, (benzoyloxy)phosphonate reductase, and benzaldehyde
decarbonylase.
- 82. The non-naturatly occurring microbial organisim of item 78, wherein said benzene
pathway comprises (benzoyl-CoA synthetase, transferase and/or hydrolase), phosphotransbenzoylase,
(benzoyloxy)phosphonate reductase, and benzaldehyde decarbonylase.
- 83. The non-naturally occurring microbial organism of item 78, wherein said benzene
pathway comprises (benzoyl-CoA synthetase, transferase and/or hydrolase), benzoyl-CoA
reductase and benzaldehyde decarboxylase.
- 84. The non-naturally occurring microbial organism of item 78, wherein said microbial
organism comprises two exogenous nucleic acids each encoding a benzene pathway enzyme.
- 85. The non-naturally occurring microbial organism of item 78, wherein said microbial
organism comprises three exogenous nucleic acids each encoding a benzene pathway enzyme.
- 86. The non-naturally occurring microbial organism of item 78, wherein said microbial
organism comprises four exogenous nucleic acids each encoding a benzene pathway enzyme.
- 87. The non-naturally occurring microbial organism of item 78, further comprising
a benzoate pathway comprising at least one exogenous nucleic acid encoding a benzoate
pathway enzyme expressed in a sufficient amount to produce benzoate, said benzoate
pathway comprising 2-dehydro-3-deoxyphosphoheptonate synthase; 3-dehydroquinate synthase;
3-dehydroquinate dehydratase; shikimate dehydrogenase; shikimate kinase; 3-phosphoshikimate-2-carboxyvinyltransferase;
chorismate synthase; and chorismate lyase.
- 88. The non-naturally occurring microbial organism of item 78, further comprising
a (2-hydroxy-4-oxobtitoxy)phosphonate pathway comprising erythrose-4-phosphate dehydratase
and (2,4-dioxobutoxy)phosphonate reductase.
- 89. The non-naturally occurring microbial organism of item 78, wherein at least one
exogenous nucleic acid is a heterologous nucleic acid.
- 90. The non-naturally occurring microbial organism of item 78, wherein said non-naturally
occurring microbial organism is in a substantially anaerobic culture medium.
- 91. A method for producing benzene, comprising culturing the non-naturally occurring
microbial organism of item 78 under conditions and for a sufficient period of time
to produce benzene.
- 92. The method of item 91, wherein said benzene pathway comprises benzoate decarboxylase.
- 93. The method of item 91, wherein said benzene pathway comprises benzoate and benzaldehyde
decarbonylase
- 94. The method of item 91, wherein said benzene pathway comprises benzoate kinase,
(benzoyloxy)phosphonate reductase, and benzaldehyde decarbonylase.
- 95. The method, of item 91, wherein said benzene pathway comprises (benzoyl-CoA synthetase,
transferase, and/or hydrolase), phosphotransbenzoylase, (benzoyloxy)phosphonate reductase,
and benzaldehyde decarbonylase.
- 96. The method of item 91, wherein said benzene pathway comprises (benzoyl-CoA synthetase,
transferase and/or hydrolase), benzoyl-CoA reductase and benzaldehyde decarbonylase.
- 97. The method of item 91, wherein said microbial organism comprises two exogenous
nucleic acids each encoding a benzene pathway enzyme.
- 98. the method of item 91, wherein said microbial organism comprises three exogenous
nucleic acids each encoding a benzene pathway enzyme.
- 99. The method of item 91, wherein said microbial organism comprises four exogenous
nucleic acids each encoding a benzene pathway enzyme.
- 100. The method of item 91, further comprising a benzoate pathway comprising at least
one exogenous nucleic acid encoding a benzoate pathway enzyme expressed in a sufficient
amount to produce benzoate, said benzoate pathway comprising 2-dehydro-3-deoxyphosphoheptonate
synthase; 3-dehydroquinate synthase; 3-dehydroquinate dehydratase; shikimate dehydrogenase;
shikimate kinase; 3-phosphoshikimate-2-carboxyvinyltransferase; chorismate synthase;
and chorismate lyase.
- 101. The method of item 91, further comprising a (2-hydroxy-4-oxobutoxy)phosphonate
pathway comprising etythrose-4-phosphate dehydratase and (2,4-dioxobutoxy)phosphonate
reductase.
- 102. The method of item 91, wherein at least one exogenous nucleic acid is a heterologous nucleic acid.
- 103. The method of item 91, wherein said non-naturally occurring microbial organism
is cultured in a substantially anaerobic culture medium.
- 104. A non-naturally occurring microbial organism, comprising a microbial organism
having a toluene pathway comprising at least one exogenous nucleic acid encoding a
toluene, pathway enzyme expressed in a sufficient amount to produce toluene, said
toluene pathway is selected from a set of pathway enzymes selected from: a) p-toluate decarboxylase; b) p-toluate reductase and p-methylbenzaldehyde decarbonylase; c) p-toluate kinase, (p-methylbenzoyloxy)phosphonate reductase, and p-methylbenzaldehyde decarbonylase; d) (p-methylbenzoyl-CoA synthetase, transferase and/or hydrolase), phosphotrans-p-methylbenzoylase, (p-methylbenzoyloxy)phosphonate reductase, and p-methylbenzaldehyde decarbonylase; and e) (p-methylbenzoyl-CoA, synthetase, transferase and/or hydrolase), p-methylbenzoyl-CoA reductase and p-methylbenzaldehyde decarbonylase, f) (p-methylbenzoyl-CoA synthetase, transferase
and/or hydrolase), p-toluate decarboxylase, g) p-methytbenzoyl-CoA reductase and p-methylbenzaldehyde
decarbonylase, h) phosphotrans-p-methylbenzoylase, (p-methylbenzoyloxy)phosphonate
reductase, and p-methylbenzaldehyde decarbonylase, i) phosphotrans-p-methylbenzoylase,
p-toluate kinase, p-toluate decarboxytase, j) phosphotrans-p-methylbenzoylase, p-toluate
kinase, p-toluate reductase, p-methylbenzaldehyde decarbonylase; k) phosphotrans-p-methylbenzoylase, (p-methylbenzoyloxy)phosphonate reductase (dephosphorylating), and p-methylbenzaldehyde decarbonylase; and 1) p-methylbenzoyl-CoA reductase and p-methylbenzaldehyde decarbonylase.
- 105. The non-naturally occuring microbial organism of item 104, wherein said toluene
pathway comprises p-toluate decarboxylase.
- 106. The non-naturally occurring microbial organism of item 104, wherein said toluene
pathway comprises p-toluate reductase and p-methylbenzaldehyde decarboxylase
- 107. The non-naturally occurring microbial orgamsm of item 104, wherein said toluene
pathway comprises c) p-toluate kinase, (p-methylbenzoyloxy)phosphonate reductase, and p-methylbenzaldehyde decarbonylase.
- 108. The non-naturally occurring microbial organism of item 104, wherein said toluene
pathway comprises (p-methytbenzoyl-CoA synthetase, transferase and/or hydrolase), phosphotrans-p-methylbenzoylase, (p-methylbenzoyloxy)phosphonate reductase, and p-methylbenzaldehyde decarbonylase.
- 109. The non-naturally occurring microbial organism of item 104, wherein said toluene,
pathway comprises (p-methylbenzoyl-CoA synthetase, transferase and/or hydrolase), p-methylbenzoyl-CoA reductase and p-methylbenzaldehyde decarbonylase.
- 110. The non-naturally occurring microbial organism of item 104, wherein said microbial
organism comprises two exogenous nucleic acids each encoding a toluene pathway enzyme.
- 111. The non-naturally occurring microbial organism of item 104, wherein said microbial
organism comprises three exogenous nucleic acids each encoding a toluene pathway enzyme.
- 112. The non-naturally occurring microbial organism of item 104, wherein said microbial
organism comprises four exogenous nucleic acids each encoding a toluene pathway enzyme.
- 113. The non-naturally occurring microbial organism of item 104, further comprising
a p-toluate pathway comprising at least one exogenous nucleic acid encoding a p-toluate pathway enzyme expressed in a sufficient amount to produce p-toluate, said p-toluate pathway comprising 2-dehydro-3-deoxyphosphoheptonate synthase; 3-dehydroquinate
synthase; 3-dehydroquinate dehydratase; shikimate dehydrogenase; shikimate kinase;
3-phosphoshikimate-2-carboxyvinyltransferase; chorismate synthase; and chorismate
lyase.
- 114. The non-naturally occurring microbial organism of item 104, further comprising a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway comprising
DXP synthase, DXP reductoisomerase, and 2ME4P dehydratase.
- 115. The non-naturally occurring microbial organism of item 104, wherein at least
one exogenous nucleic acid is a heterologous nucleic acid.
- 116. The non-naturally occurring microbial organism of item 104, wherein said non-naturally
occurring microbial organism is in a substantially anaerobic culture medium.
- 117. A method for producing toluene, comprising culturing the non-naturally occurring
microbial organism of item 104 under conditions and for a sufficient period of time to produce toluene.
- 118. The method of item 117, wherein said toluene pathway comprises p-toluate decarboxylase.
- 119. The method of item 117, wherein said toluene pathway comprises p-toluate reductase and p-methylbenzaldehyde decarbonylase
- 120. The method of item 117, wherein said toluene pathway comprises c) p-toluate kinase, (p-methylbenzoyloxy)phosphonate reductase, and p-methylbenzaldehyde decarbonylase.
- 121. The method of item 117, wherein said toluene pathway comprises (p-methylbenzoyl-CoA synthetase, transferase and/or hydrolase), phosphotrans-p-methylbenzoylase, (p-methylbenzoyloxy)phosphonate reductase, and p-methylbenzaldehyde decarbonylase,
- 122. The method of item 117, wherein said toluene pathway comprises (p-methylbenzoyl-CoA synthetase, transferase and/or hydrolase), p-methylbenzoyl-CoA reductase and p-methylbenzaldehyde decarbonylase.
- 123. The method of item 117, wherein said microbial organism comprises two exogenous
nucleic acids each encoding a toluene pathway enzyme.
- 124. The method of item 117, wherein said microbial organism comprises three exogenous
nucleic acids each encoding a toluene pathway enzyme.
- 125. The method of item 117, wherein said microbial organism comprises four exogenous
nucleic acids each encoding a toluene pathway enzyme.
- 126. The method of item 117, further comprising a p-toluate pathway comprising at least one exogenous nucleic acid encoding a p-toluate pathway enzyme expressed in a sufficient amount to produce p-toluate, said p-toluate pathway comprising 2-dehydro-3-deoxyphosphoheptonate synthase; 3-dehydroquinate
synthase; 3-dehydroquinate dehydratase; shikimate dehydrogenase; shikimate kinase;
3-phosphoshikimate-2-carboxyvinyltransferase; chorismate synthase; and chorismate
lyase.
- 127. The method of item 117, further comprising a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate
pathway comprising DXP synthase, DXP reductoisomerase, and 2ME4P dehydratase,
- 128. The method of item 117, wherein at least one exogenous nucleic acid is a heterologous
nucleic acid.
- 129. The method of item 117, wherein, non-naturally occurring microbial is cultured
in a substantially anaerobic culture medium.
- 130. A non-naturally occurring microbial organism, comprising a microbial organism
having a 2,4-pentadienoate pathway comprising at least one exogenous nucleic acid
encoding a 2,4-pentadienoate pathway enzyme expressed in a sufficient amount to produce
2,4-pentadienoate, 2,4-pentadienoate pathway having a set of enzymes selected from
A) i) a 4-hydroxy-2-oxovaterate aldolase, ii) a 4-hydroxy-2-oxovalerate dehydratase,
iii) a 2-oxopentenoate reductase, and iv) a 2-hydroxypentenoate dehydratase; B) i)
an AKP deaminase, ii) an acetylacrylate reductase, and iii) a 4-hydroxypent-2-enoate
dehydratase; C) i) an AKP aminotransferase and/or dehydrogenase, ii) a 2,4-dioxopentanoate-2-reductase,
iii) a 2-hydroxy-4-oxopentanoate dehydratase, iv) an acetylacrylate reductase, and
v) a 4-hydroxypent-2-enoate dehydratase; D) i) an AKP aminotransferaseand/or dehydrogenase,
ii) a 2,4-dioxopentanoate-4-reductase, iii) a 4-hydroxy-2-oxovalerate dehydratase,
and E), iv) a 2-oxopentenoate reductase, and v) a 2-hydroxypentenoate dehydratase;
and E) i) an AKP reductase, ii) a 2-amino-4-hydroxypentanoate aminotransferase and/or
dehydrogenase, iii) a 4-hydroxy-2-oxovalerate dehydratase, iv) a 2-oxopentenoate reductase,
and v) a 2-hydroxypentenoate dehydratase.
- 131. The non-naturally occurring microbial organism of item 130, wherein said microbial
organism comprises two exogenous nucleic acids each encoding a 2,4-pentadienoate,
pathway enzyme.
- 132. The non-naturally occurring microbial organism of item 130, wherein said microbial
organism comprises three exogenous nucleic acids each encoding a 2,4-pentadienoate
pathway enzyme.
- 133. The non-naturally occurring microbial organism of item 132, wherein said three
exogenous nucleic acids encode i) an AKP deaminase, ii) an acetylacrylate reductase,
iii) a 4-hydroxypent-2-enoate dehydratase.
- 134. The non-naturally occurring microbial organism of item 130, wherein said microbial
organism comprises four exogenous nucleic acids each encoding a 2,4-pentadienoate
pathway enzyme.
- 135. The non-naturally occurring microbial organism of item 134, wherein said four
exogenous nucleic acids encode i) a 4-hydroxy-2-oxovaterate aldolase, ii) a 4-hydroxy-2-oxovalerate
dehydratase, iii) a 2-oxopentenoate reductase, and iv) a 2-hydroxypentenoate dehydratase.
- 136. The non-naturally occurring microbian organism of item 130, wherein said microbial
organism comprises five exogenous nucleic acids each encoding a 2,4-pentadienoate,
pathway enzyme.
- 137. The non-naturally occurring microbial organism of item 136, wherein said five
exogenous nucleic acids encode i) an AKP aminotransferase and/or dehydrogenase, ii)
a 2,4-dioxopentanoate-2-reductase, iii) 2-hydroxy-4-oxopentanoate dehydratase, iv)
an acetylacrylate reductase, and v) a 4-hydroxypent-2-enoate dehydratase.
- 138. The non-naturally occurring microbial organism of item 136, wherein said five
exogenous nucleic acids encode i) a AKP aminotransferase and/or dehydrogenase, ii)
a, 2,4-dioxopentanoate-4-reductase, iii) a 4-hydroxy-2-oxovalerate dehydratase, iv)
a 2-oxopentenoate reductase, and v) a 2-hydroxypentenoate dehydratase.
- 139. The non-naturally occurring microbial organism of item 136, wherein said five
exogenous nucleic acids encode i) an AKP reductase, ii) a 2-amino-4-hydroxypentanoate
aminotransferase and/or dehydrogenase, iii) a 4-hydroxy-2-oxovalerate dehydratase,
iv) a 2-oxopentenoate and v) a 2-hydroxypentenoate dehydratase.
- 140. The non-naturally occurring microbial organism of item 130, wherein said at least
one exogenous nucleic acid is a heterologous nucleic acid.
- 141. The non-naturally occurring microbial organism of item 130, wherein, said non-naturally
occurring microbial organism is in a substantially culture medium.
- 142. The non-naturally occurring microbial organism of item 130, further comprising
a 2,4-pentadienoate decarboxylase expressed in a sufficient amount to produce 1,3-butadiene
by conversion of 2,4-pentadienoate to 1,3-butadiene.
- 143. The non-natturally occurring microbial organism of item 130, further comprising
at least one of an AKP thiolase, an ornithine 4,5-aminomutase, a 2,4-diaminopentanoate
4-aminotransferase and a 2,4-diaminopentanoate 4-dehydrogenase.
- 144. A method for producing 2,4-pentadienoate, comprising culturing a non-naturally
occurring microbial organism having a 2,4-pentadienoate pathway, said pathway comprising
at least exogenous nucleic acid encoding a 2,4-pentadienoate pathway enzyme expressed
in a sufficient amount to produce 2,4-pentadienoate, under conditions and for a sufficient
period of time to produce 2,4-pentadienoate, said 2,4-petitadienoate pathway selected
from A) i) a 4-hydroxy-2-oxovalerate aldolase, ii) a 4-hydroxy-2-oxovalerate dehydratase,
iii) a 2-oxopentenoate reductase, and iv) a 2-hydroxypentenoate dehydratase, B) i)
an AKP deaminase, ii) an acetylacrylate reductase, and iii) a 4-hydroxypent-2-enoate
dehydratase; C) i) an AKP aminotransferase and/or dehydrogenase, ii) a 2,4-dioxopentanoate-2-reductase,
iii) a 2-hydroxy-4-oxopentanoate dehydratase, iv) an acetytacrylate reductase, and
v) a 4-hydroxypent-2-enoate dehydratase; D) i) an AKP aminotransferase and/or dehydrogenase,
ii) a 2,4-dioxopentanoate-4-reductase, iii) a 4-hydroxy-2-oxovalerate dehydratase,
iv) a 2-oxopentenoate reductase, and v) a 2-hydroxypentenoate dehydratase; and E)
i) an AKP reductase, ii) a 2-amino-4-hydroxypentanoate aminotransferase and/or dehydrogenase,
iii) a 4-hydroxy-2-oxovalerate dehydratase, iv) a 2-oxopentenoate reductase, and v)
a 2-hydroxypentenoate dehydratase,
- 145. The method of item 144, wherein said non-naturally occurring microbial organism
is in a substantially anaerobic culture medium.
- 146. The method of item 144, wherein said at least one exogenous nucleic acid is a
heterologous nucleic acid.
- 147. The method of item 144, wherein said microbial organism comprises two exogenous
nucleic acids each encoding a 2,4-pentadienoate pathway enzyme.
- 148. The method of item 144, wherein said microbial organism comprises three exogenous
nucleic acids each encoding a 2,4-pentadienoate pathway enzyme.
- 149. The method of item 148, wherein said three exogenous nucleic acids encode i)
an AKP deaminase, ii) an acetylacrylate reductase, and iii) a 4-hydroxypent-2-enoate
dehydratase.
- 150. The method of item 144, wherein said microbial organism, comprises four exogenous
nucleic acids each encoding a 2,4-pentadienoate pathway enzyme.
- 151. The method, of item 150, wherein said four exogenous nucleic acids encode i)
a 4-hydroxy-2-oxovalerate aldolase, ii) a 4-hydroxy-2-oxovalerate dehydratase, iii)
a 2-oxopentenoate reductase, and iv) a 2-hydroxypentenoate dehydratase.
- 152. The method of item 144, wherein said microbial organism comprises five exogenous
nucleic acids each encoding a 2,4-pentadienoate pathway enzyme.
- 153. The method, of item 152, wherein said five exogenous nucleic acids encode i)
an AKP aminotransferase and/or dehydrogenase, ii) a 2,4-dioxopentanoate-2-reductase,
iii) a 2-hydroxy-4-oxopentanoate dehydratase, iv) an acetylacrylate reductase, and
v) a 4-hydroxypent-2-enoate dehydratase.
- 154. The method of item 152, wherein said five exogenous nucleic acids encode i) an
AKP aminotransferase and/or dehydrogenase, ii) a 2,4-dioxopentanoate-4-reductase,
iii) a 4-hydroxy-2-oxovalerate dehydratase, iv) a 2-oxopentenoate reductase, and v)
a 2-hydroxypentenoate dehydratase.
- 155. The method of item 152, wherein said five exogenous nucleic acids encode i) an
AKP reductase, ii) a 2-amino-4-hydroxypentanoate aminotransferase and/or dehydrogenase,
iii) a 4-hydroxy-2-oxovalerate dehydratase, iv) a 2-oxopentenoate reductase, and v)
a 2-hydroxypentenoate dehydratase.
- 156. The method of item 144, further comprising a 2,4-pentadienoate decarboxylase
expressed in a sufficient amount to convert 2,4-pentadienoate to 1,3-butadiene.
- 157. The method of item 144, further comprising at least one of an AKP thiolase, an
ornithine 4,5-aminomutase, a 2,4-diaminopentanoate 4-aminotransferase a 2,4-diaminopentanoate
4-dehydrogenase.
- 158. A non-naturally occurring microbial organism, comprising a microbial organism
having a 2,4-pentadienoate pathway comprising at least one exogenous nucleic encoding
a 2,4-pentadienoate pathway enzyme expressed in a sufficient amount to produce 2,4-pentadienoate,
2,4-pentadienoate pathway having a set of enzymes selected from:
1) A. 3-hydroxypiopanoyl-CoA acetyltratisferase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase,
C. 3,5-dihydroxypentanoyl-CoA dehydratase, D. 5-hydroxypent-2-enoyl-CoA dehydratase,
E. pent-2,4-dienoyl-CoA synthetase, transferase and/or hydrolase;
2) A. 3-hydroxypropanoyl-CoA acetyltransferase, B. 3-oxo-5-hydroxypentanoyl-COA reductase,
G. 3,5-dihydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, J. 3,5-dihydroxypentanoate
dehydratase, H. 5-hydroxypent-2-enoyl-CoA synthetase, transferase and/or hydrolase,
D. 5-hydroxypent-2-enoyl-CoA dehydratase, E. pent-2,4-dienoyl-CoA synthetase, and/or
hydrolase;
3) A. 3-hydroxypropanoyl-CoA acetyltransferase, F. 3-oxo-5-hydroxypentanoyl-CoA synthetase,
and/or hydrolase, I. 3-oxo-5-hydroxypentatioate reductase, G. 3,5-dihydroxypentanoyl-CoA
synthetase, transferase and/or hydrolase, C. 3,5-dihydroxypentanoyl-CoA dehydratase,
D. 5-hydroxypent-2-enoyl-CoA dehydratase, E. pent-2,4-dienoyl-CoA synthetase, transferase
and/or hydrolase;
4) A. 3-hydroxypropanoyl-CoA acetyltransferase, F. 3-oxo-5-hydroxypentanoyl-CoA, synthetase,
transferase and/or hydrolase, I. 3-oxo-5-hydroxypentanoate reductase, J. 3,5-dihydroxypentanoate
dehydratase, H. 5-hydroxypent-2-enoyl-CoA synthetase, transferase and/or hydrolase,
D. 5-hydroxypent-2-enoyl-CoA dehydratase, E. pent-2,4-dienoyl-CoA synthetase, transferase
and/or hydrotase;
5) K. 3-hydroxypropanoyl-COA dehydratase, A. 3-hydroxypropanoyl-CoA acetyltransferase,
B. 3-oxo-5-hydroxypentanoyl-CoA reductase, C. 3,5-dihydroxypentanoyl-CoA dehydratase,
D. 5-hydroxypent-2-enoyl-CoA dehydratase, E. pent-2,4-dienoyl-CoA synthetase, transferase
and/or hydrolase;
6) K. 3-hydroxypropanoyl-CoA dehydratase, A. 3-hydroxypropanoyl-CoA acetyltransferase,
B. 3-oxo-5-hydroxypentanoyl-CoA reductase, G. 3,5-dihydroxypentanoyl-CoA synthetase,
transferase, and/or hydrolase, J. 3,5-dihydroxypentanoate dehydratase, H. 5-hydroxypent-2-enoyl-CoA
synthetase, transferase and/or hydrolase, D. 5-hydroxypent-2-enoyl-CoA dehydratase,
E. pent-2,4-dienoyl-CoA synthetase, transferase and/or hydrolase;
7) K. 3-hydroxypropanoyl-CoA dehydratase, A. 3-hydroxypropanoyl-CoA acetyltransterase,
F. 3-oxo-5-hydroxypertanoyl-CoA synthetase, and/or hydrolase, 1. 3-oxo-5-hydroxypentanoate
reductase, G. 3,5-d.ihydroxypentanoyl-CoA synthetase, transferase and/or hydrolase,
C. 3,5-dihydroxypentanoyl-CoA dehydratase, D. 5-hydroxypent-2-enoyl-CoA dehydratase,
E. pent-2,4-dienoyl-CoA synthetase, transferase and/or hydrolase;
8) K. 3-hydroxypropanoyl-CoA dehydratase, A. 3-hydroxypropanoyl-CoA acetyltransferase,
F. 3-oxo-5-hydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, 1. 3-oxo-5-hydroxypentanoate
reductase, J. 3,5-dihydroxypentanoate dehydratase, H. 5-hydroxypent-2-enoyl-CoA synthetase,
transferase and/or hydrolase, D. 5-hydroxypent-2-enoyl-CoA dehydratase, E. pent-2,4-dienoyl-CoA
synthetase, and/or hydrolase;
9) M. acrylyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase,
B. 3-oxo-5-hydroxypentanoyl-CoA reductase, C. 3,5-dihydroxypentanoyl-CoA dehydratase,
D. 5-hydroxypent-2-enoyl-CoA dehydratase, E. pent-2,4-dienoyl-CoA synthetase, transferase
and/or hydrolase;
10) M. acrylyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanol-CoA dehydratase,
B. 3-oxo-5-hydroxypentanoyl-CoA reductase, G. 3,5-dihydroxypentanoyl-CoA synthetase,
transferase and/or hydrolase, J. 3,5-dihydroxypentanoate dehydratase, H. 5-hydroxypent-2-enoyl-CoA
synthetase, transferase and/or hydrolase, D. 5-hydroxypent-2-enoyl-CoA dehydratase,
E. pent-2,4-dienoyl-CoA synthetase, transferase and/or hydrolase;
11) M. acrylyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase,
F. 3-oxo-5-hydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, I. 3-oxo-5-hydroxypentanoate
reductase, G. 3,5-dihydroxypentanoyl-CoA synthetase, transferase and/or hydrolase,
C. 3,5-dihydroxypentanoyl-CoA dehydratase, D. 5-hydroxypent-2-enoyl-CoA dehydratase,
E. pent-2,4-dienoyl-CoA synthetase, transferase and/or hydrolase;
12) M. acrylyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase,
, F. 3-oxo-5-hydroxypentanoyl-CoA synthetase, and/or hydrolase, I. 3-oxo-5-hydroxypentanoate
reductase, J. 3,5-dihydroxypentanoate dehydratase, H. 5-hydroxypent-2-enoyl-CoA synthetase,
transferase and/or hydrolase, D. 5-hydroxypent-2-enoyl-CoA dehydratase, E. pent-2,4-dienoyl-CoA
synthetase, transferase and/or hydrolase;
13) M. acrylyl-CoA acetyltransferase, N. 3-oxopent-4-enoyl-CoA reductase, R. 3-hydroxypent-4-enoyl-CoA
dehydratase, E. pent-2,4-dienoyl-CoA synthetase, transferase and/or hydrolase;
14) M. acrylyl-CoA acetyltransferase, N. 3 -oxopent-4-enoyl-CoA reductase, T. 3-hydroxypent-4-enoyl-CoA
transferase, synthetase or hydrolase, S. 3-hydroxypent-4-enoate dehydratase; and
15) M. acrylyl-CoA acetyltransferase, O. 3-oxopent-4-enoyl-COA synthetase, transferase,
and/or hydrolase, P. 3-oxopent-4-enoate reductase, S. 3-hydroxypent-4-enoate dehydratase;
16) A. 3-hydroxypropanoyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase,
N. 3-oxopent-4-enoyl-CoA reductase, R. 3-hydroxypent-4-enoyl-CoA dehydratase, E. pent-2,4-dienoyl-CoA
synthetase, transferase and/or hydrolase;
17) A. 3-hydroxypropanoyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase,
N. 3-oxopent4-enoyl-CoA reductase, T. 3-hydroxypent-4-enoyt-CoA transferase, synthetase,
or hydrolase, S. 3-hydroxypent-4-enoate dehydratase;
18) A. 3-hydroxypropanoyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase,
O.3-oxopent-4-enoyl-CoA synthetase, transferase and/or hydrolase, P. 3-oxopent-4-enoate
reductase, S. 3-hydroxypent-4-enoate dehydratase.
19) A. 3-hydroxypropanoyl-CoA acetyltransferase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase,
C. 3,5-dihydroxypentanoyl-CoA dehydratase, H.5-hydroxypent-2-enoyl-CoA synthetase,
transferase and/or hydrolase, Q. 5-hydroxypent-2-enoate dehydratase;
20) A. 3-hydroxypropanoyl-CoA acetyltransferase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase,
G. 3,5-dihydroxypentanoyl-CoA synthetase, and/or hydrolase, J. 3,5-dihydroxypentanoate
dehydratase, Q. 5-hydroxypent-2-enoate dehydratase;
21) A. 3-hydroxypropanoyl-CoA acetyltransferase, F. 3-oxo-5-hydroxypentanoyl-CoA synthetase,
transferase and/or hydrolase I. 3-oxo-5-hydroxypentanoate reductase, J. 3,5-dihydroxypentanoate
dehydratase, Q. 5-hydroxypent-2-enoate dehydratase;
22) M. acrylyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase,
F. 3-oxo-5-hydroxypentanoyl-CoA synthetase, transferase and/or hydrolase 1. 3-oxo-5-hydroxypentanoate
reductase, J. 3,5-dihydroxypentanoate dehydratase, Q. 5-hydroxypent-2-enoate dehydratase;
23) M. acrylyl-CoA. acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase,
B. 3-oxo-5-hydroxypentanoyl-CoA reductase, G. 3,5-dihydroxypentanoyl-CoA synthetase,
transferase and/or hydrolase, J. 3,5-dihydroxypentanoate dehydratase, Q. 5-hydroxypent-2-enoate
dehydratase; and
24) M. acrylyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase,
B. 3-oxo-5-hydroxypentanoyl-CoA reductase, C. 3,5-dihydroxypentanoyl-CoA dehydratase,
H. 5-hydroxypent-2-enoyl-CoA synthetase, transferase and/or hydrolase, Q. 5-hydroxypent-2-enoate
dehydratase.
- 159. The non-naturally occurring microbial organism of item 158, wherein said microbial
organism comprises two, three, four, five, six, seven, or eight exogenous nucleic
acids each encoding a 2,4-pentadienoate pathway enzyme.
- 160. The non-naturally occurring microbial organism of item 158, wherein said at least
one exogenous nucleic acid is a heterologous nucleic acid.
- 161. The non-naturally occurring microbial organism of item 158, wherein said non-naturally
occurring microbial organism is in a substantially anaerobic culture medium.
- 162. The non-naturally occurring microbial organism of item 158, further comprising
a 2,4-pentadiene decarboxylase to convert 2,4-pentadienoate to 1,3-butadiene.
- 163. A method for producing 2,4-pentadienoate, comprising culturing a non-naturally
occurring microbial organism according to item 158, under conditions and for a sufficient
period of time to produce 2,4-pentadienoate.
- 164. The method of item 163, wherein said microbial organism comprises two, three,
four, five, six, seven, or eight exogenous nucleic acids each encoding a 2,4-pentadienoate
pathway enzyme.
- 165. The method of item 163, wherein said at least one exogenous nucleic acid is a
heterologous nucleic acid.
- 166. The method of item 163, wherein said non-naturally occurring microbial organism
is in a substantially anaerobic culture medium.
- 167. A method for producing 1,3-butadiene, comprising culturing a non-naturally occurring
microbial organism according to item 162, under conditions and for a sufficient period
of time to produce 1,3-butadiene.
- 168. The method of item 167, wherein said microbial organism comprises two, three,
four, five, six, seven, or eight exogenous nucleic acids each encoding a 2,4-pentadienoate
pathway enzyme.
- 169. The method of item 167, wherein said at least one exogenous nucleic acid is a
heterologous nucleic acid.
- 170. The method of item 167, wherein said non-naturally occurring microbial organism
is in a substantially anaerobic culture medium.
- 171. A non-naturally occurring microbial organism, comprising a microbial organism
having a 1,3-butadiene pathway comprising at least one exogenous nucleic acid encoding
a 1,3-butadiene pathway enzyme expressed in a sufficient amount to produce 1,3-butadiene,
said 1,3-butadiene pathway having a set of enzymes selected from:
- 1) M. acrylyl-CoA acetyltransferase, N. 3-oxopent-4-enoyl-CoA reductase, T. 3-hydioxypent-4-enoyl-CoA
transferase, synthetase or hydrolase, Y. 3-hydroxypent-4-enoate decarboxylase;
- 2) M. acrylyl-CoA acetyltransferase, O. 3-oxopent-4-enoyl-CoA synthetase, transferase
and/or hydrolase, P. 3-oxopent-4-enoate reductase, Y. 3-hydroxypent-4-enoate decarboxylase;
- 3) K. 3-hydroxypropanoyl-CoA dehydratase, M. acrylyl-CoA acetyltransferase, N. 3-oxopent-4-enoyl-CoA
reductase, T. 3-hydroxypent-4-enoyl-CoA transferase, synthetase or hydrolase, Y. 3-hydroxypent-4-onoate
decarboxylase-,
- 4) K. 3-hydroxypropanoyl-CoA dehydratase, M. acrylyl,-CoA acetyltransferase, O. 3-oxopent-4-enoyl-CoA
synthetase, transferase and/or hydrolase, P. 3-oxopent-4-enoate reductase, Y. 3-hydroxypent-4-enoate
decarboxylase;
- 5) A. 3-hydroxypropanoyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase,
N. 3-oxopent-4-enoyl-CoA reductase, T. 3-hydroxypent-4-enoyl-CoA transferase, synthetase
or hydrolase, Y. 3-hydroxypent-4-enoate decarboxylase;
- 6) A. 3-hydroxypropanoyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-COA dehydratase,
O. 3-oxopent-4-enoyl-CoA synthetase, transferase and/or hydrolase, P. 3-oxopent-4-enoate
reductase, Y. 3-hydroxypent-4-enoate decarboxylase;
- 172. The non-naturally occurring microbial organism of item 171, wherein said microbial
organism comprises two, three, four, or five exogenous nucleic acids each encoding
a 1,3-butadiene pathway enzyme.
- 173. The non-naturally occurring microbial organism of item 171, wherein, said at
least one exogenous nucleic acid is a heterologous nucleic acid.
- 174. The non-naturally occurring microbial organism of item 171, wherein said non-naturally
occurring microbial organism, is in a substantially anaerobic culture medium.
- 175. A method for producing 1,3-butadiene, comprising culturing a non-naturally occurring
microbial organism, according to item 171, under conditions and for a sufficient period
of time to produce 1,3-butadiene.
- 176. The method of item 175, wherein said microbial organism comprises two, three,
four, or five exogenous nucleic acids each encoding a 1,3-butadiene pathway enzyme.
- 177. The method of item 175, wherein said at least one exogenous nucleic acid is a
heterologous nucleic acid.
- 178. The method of item 175, wherein said non-naturally occurring microbial organism
is in a substantially anaerobic culture medium.
- 179. A non-naturally occurring microbial organism, comprising a microbial organism
having a 1,3-butadiene pathway comprismg at least one exogenous nucleic acid encoding
a 3-butene-1-ol pathway enzyme expressed in a sufficient amount to produce 3-butene-1-ol,
said 3-butene-1-ol pathway having a set of enzymes selected from:
1) A. 3-hydroxypropanoyl-CoA acetyltransferase, F. 3-oxo-5-hydroxypentanoyl-CoA synthetase,
transferase and/or hydrolase, 1.3-oxo-5-hydroxypentanoate reductase, U. 3,5-dihydroxypentanoate
decarboxylase;
2) A. 3-hydroxypropanoyl-CoA acetyltransferase, F. 3-oxo-5-hydroxypentanoyl-CoA synthetase,
transferase and/or hydrolase, I: 3-oxo-5-hydroxypentanoate reductase, J. 3,5-dihydroxypentanoate
dehydratase, V. 5-hydroxypent-2-enoate decarboxylase;
3) A. 3-hydroxypropanoyl-CoA acetyltransferase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase,
G. 3,5-dihydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, U. 3,5-dihydroxypentanoate
decarboxylase;
4) A. 3-hydroxypropanoyl-CoA acetyltransferase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase,
G. 3,5-dihydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, J. 3,5-dihydroxypentanoate
dehydratase, V. 5-hydroxypent-2-enoate decarboxylase;
5) A. 3-hydroxypropanoyl-CoA acetyltransferase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase,
C. 3,5-dihydroxypentanoyl-CoA dehydratase, H. 5-hydroxypent-2-enoyl-CoA synthetase,
transferase and/or hydrolase, V. 5-hydroxypent-2-enoate decarboxylase;
6) M. acrylyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase,
F. 3-oxo-5-hydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, I. 3-oxo-5-hydroxypentanoate
reductase, U. 3,5-dihydroxypentanoate decarboxylase;
7) M. acrylyl-COA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase,
F. 3-oxo-5-bydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, I. 3-oxo-5-hydroxypentanoate
reductase, J. 3,5-dihydroxypentanoate dehydratase, V. 5-hydroxypetit-2-enoate decarboxylase;
8) M. acrylyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase,
B. 3-oxo-5-hydroxypentanoyl-CoA reductase, G. 3,5-dihydroxypentanolyl-CoA synthetase,
transferase and/or hydrolase, U. 3,5-dihydroxypentanoate decarboxylase;
9) M. acrylyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase,
B. 3-oxo-5-hydroxypentanoyl-CoA reductase, G. 3,5-dihydroxypentanoyl-CoA synthetase,
transferase and/or hydrolase, J. 3,5-dihydroxypentanoate dehydratase, V. 5-hydroxypent-2-enoate
decarboxylase;
10) M. acrylyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase,
B. 3-oxo-5-hydroxypentanoyl-CoA reductase, C. 3,5-dihydroxypentanoyl-CoA dehydratase,
H. 5-hydroxypent-2-enoyl-CoA synthetase, transferase and/or hydrolase, V. 5-hydroxypent-2-enoate
decarboxylase;
11) K. 3-hydroxypropanoyl-CoA dehydratase, A. 3-hydroxypropanoyl-CoA. acetyltransferase,
F. 3-oxo-5-hydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, I. 3-oxo-5-hydroxypentanoate
reductase, U. 3,5-dihydroxypentanoate decarboxylase;
12) K. 3-hydroxypropanoyl-CoA dehydratase, A. 3-hydroxypropanoyl-CoA acetyltransferase,
F. 3-oxo-5-hydroxypentanoyl-CoA synthetase, and/or hydrolase, I. 3-oxo-5-hydroxypentanoate
reductase, J. 3,5-dihydroxypentanoate dehydratase, V. 5-hydroxypent-2-enoate decarboxylase;
13) K. 3-hydroxypropanoyl-CoA dehydratase, A. 3-hydroxypropanoyl-CoA acetyltransferase,
B. 3-oxo-5-hydroxypentanoyl-CoA reductase, G. 3,5-dihydroxypentanoyl-CoA synthetase,
transferase and/or hydrolase, U. 3,5-dihydroxypentanoate decarboxylase;
14) K. 3-hydroxypropanoyl-CoA dehydratase, A. 3-hydroxypropanoyl-CoA acetyltransferase,
B. 3-oxo-5-hydroxypentanoyl-CoA reductase, G. 3,5-dihydroxypentanoyl-CoA synthetase,
transferase and/or hydrolase, J. 3,5-dihydroxypentanoate dehydratase, V. 5-hydroxypent-2-enoate
decarboxylase;
15) K. 3-hydroxypropanoyl-CoA dehydratase, A. 3-hydroxypropanoyl-CoA acetyltransferase,
B. 3-oxo-5-hydroxypentanoyl-CoA reductase, C. 3,5-dihydroxypentanoyl-CoA dehydratase,
H. 5-hydroxypent-2-enoyl-CoA synthetase, transferase and/or bydrotase, V. 5-hydroxypent-2-enoate
decarboxylase;
16) K. 3-hydroxypropanoyl-CoA dehydratase, M. acrylyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA
dehydratase, F. 3-oxo-5-hydroxypentanoyl-CoA synthetase, transferase and/or hydrolase,
1. 3-oxo-5-hydroxypentanoate reductase, U. 3,5-dihydroxypentanoate decarboxylase;
17) K. 3-hydroxypropanoyl-CoA dehydratase, M. acrylyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA
dehydratase, F. 3-oxo-5-hydroxypentanoyl-CoA synthetase, transferase and/or hydrolase,
I. 3-oxo-5-hydroxypentanoate reductase, J. 3,5-dihydroxypentanoate dehydratase, V.
5-hydroxypent-2-enoate decarboxylase;
18) K. 3-hydroxypropanoyl-CoA dehydratase, M. acrylyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA
dehydratase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase, G. 3,5-dihydroxypentanoyl-CoA
synthetase, transferase and/or hydrolase, U. 3,5-dihydroxypentanoate decarboxylase;
19) K. 3-hydroxypropanoyl-CoA dehydratase, M. acrylyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA
dehydratase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase, G. 3,5-dihydroxypentanoyl-CoA
synthetase, transferase and/or hydrolase, J. 3,5-dihydroxypentanoate dehydratase,
V. 5-hydroxypent-2-enoate decarboxylase;
20) K. 3-hydroxypropanoyl-CoA dehydratase, M. acrylyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA
dehydratase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase, C. 3,5-dihydroxypentanoyl-CoA
dehydratase, H. 5-hydroxypent-2-enoyl-CoA synthetase, transferase and/or hydrolase,
V. 5-hydroxypent-2-enoate decarboxylase;
- 180. The non-naturally occurring microbial organism of item 179, wherein said microbial
organism comprises two, three, four, five, six, or seven, exogenous nucleic acids
each encoding a 3-butene-1-ol pathways enzyme.
- 181. The non-naturally occurring microbial organism of item 179, wherein said at least
one exogenous nucleic acid is a heterologous nucleic acid.
- 182. The non-naturally occurring microbial organism of item 179, wherein said non-naturally
occurring microbial organism is in a substantially anaerobic culture medium.
- 183. The non-naturally occurring microbial organism of item 179, further comprising
a 3-butene-1-ol dehydratase to convert 3-butene-1-ol to 1,3-butadiene.
- 184. A method for producing 3-butene-1-ol, comprising culturing a non-naturally occurring
microbial organism according to item 179, under conditions and for a sufficient period
of time to produce 3-butene-1-ol.
- 185. The method of item 184, wherein said microbial organism comprises two, three,
four, five, six, or seven exogenous nucleic acids each encoding a 3-butene-1-ol pathways
enzyme.
- 186. The method of item 184, wherein said at least one exogenous nucleic acid is a
heterologous nucleic acid.
- 187. The method of item 184, wherein said non-naturally occurring microbial organism
is in a substantially anaerobic culture medium.
- 188. The method of item 184 further comprising the chemical dehydration of 3-butene-1-ol
to provide 1,3-butadiene.
- 189. A method for producing 1,3-butadiene, comprising culturing a non-naturally occurring
microbial organism according to item 183, under conditions and for a sufficient period
of time to produce 1,3-butadiene.
- 190. The method of item 189, wherein said microbial organism comprises two, three,
four, five, six, seven, or exogenous nucleic encoding a 1,3-butadiene pathway, enzyme.
- 191. The method of item 189, wherein said at least one exogenous nucleic acid is a
heterologous nucleic acid.
- 192. The method of item 189, wherein said non-naturally occurring microbial organism
is in a substantially anaerobic culture medium.
- 193. A non-naturally occurring microbial organism, comprising a microbial organism
having a 1,3-butadiene pathway comprising at least one exogenous nucleic acid encoring
a 1,3-butadiene pathway enzyme expressed in a sufficient amount to produce 1,3-butadiene,
said 1,3-butadiene pathway selected from:
(A) a succitiyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA transferase, a 3-oxoadipyl-CoA
synthetase or a 3-oxoadipyl-CoA hydrolase; a 3-oxoadipate dehydrogenase; a 2-fumarylacetate
decarboxylase; a 3-oxopent-4-enoate reductase; and a 3-hydroxypent-4-enoate decarboxylase;
(B) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA transferase, a 3-oxoadipyl-CoA
synthetase or a 3-oxoadipyl-CoA hydrolase; a 3-oxoadipate dehydrogenase; a 2-fumarylacetate
decarboxylase; a 3-oxopent-4-enoate reductase; a 3-hydroxypent-4-enoatc dehydratase;
and a 2,4-pentadienoate decarboxylase;
(C) a suceinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA transferase, a 3-oxoadipyl-CoA
synthetase or a 3-oxoadipyl-CoA hydrolase; a 3-oxoadipate dehydrogenase; a 2-fumarylacetate
reductase; a 3-hydroxyhex-4-enedioate decarboxylase; and a 3-hydroxypent-4-etioate
decarboxylase;
(D) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA transferase, a 3-oxoadipyl-CoA.
synthetase or a 3-oxoaiipyl-CoA hydrolase; a 3-oxoadipate dehydrogenase; a 2-fumarylacetate
reductase; a 3-hydroxyhex-4-enedioate decarboxylase; a 3-hydroxypent-4-enoate dehydratase;
and a 2,4-pentadienoate decarboxylase;
(E) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA transferase, a 3-oxoadipyl-CoA
synthetase or a 3-oxoadipyl-CoA hydrolase; a 3-oxoadipate reductase; a 3-hydroxyadipate
dehydrogenase; a 3-hydroxyhex-4-enedioate decarboxylase; and a 3-hydroxypent-4-enoate
decarboxylase;
(F) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA transferase, a 3-oxoadipyl-CoA
synthetase or a 3-oxoadipyl-CoA hydrolase; a 3-oxoadipate reductase; a 3-hydroxyadipate
dehydrogenase; a 3-hydroxyhex-4-enedioate decarboxylase; a 3-hydroxypent-4-enoate
dehydratase; and a 2,4-pentadienoate decarboxylase;
(G) a succinyl-CoA:acetyl-CoA acyl transferase; a 3-oxoadipyl-CoA reductase; a 3-hydroxyadipyt-CoA
transferase, a 3-hydroxyadipyl-CoA synthetase or a 3-hydroxyadipyl-CoA hydrolase;
a 3-hydroxyadipate dehydrogenase; a 3-hydroxyhex-4-enedioate decarboxylase; and a
3-hydroxypent-4-enoate decarboxylase; and
(H) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA. reductase; a 3-hydroxyadipyl-CoA
transferase, a 3-hydroxyadipyl-CoA synthetase or a 3-hydroxyadipyl-CoA hydrolase;
a 3-hydroxyadipate dehydrogenase; a 3-hydroxyhex-4-enedioate decarboxylase; a 3-hydroxypent-4-enoate
dehydratase; and a 2,4-pentadienoate decarboxylase.
- 194. The non-naturally occurring microbial organism of item 193, wherein said, microbial
organism comprises two, three, four, five, six or seven, exogenous nucleic acids each
encoding a 1,3-butadiene pathway enzyme.
- 195. The non-naturally occurring microbial organism of item 194, wherein said microbial
organism comprises exogenous nucleic acids encoding each of the enzymes selected from:
(A) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA transferase, a 3-oxoadipyl-CoA
synthetase or a 3-oxoadipyl-CoA hydrolase; a 3-oxoadipate dehydrogenase; a 2-fumarylacetate
decarboxylase; a 3-oxopent-4-enoate reductase; and a 3-hydroxypent-4-enoate decarboxylase;
(B) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA transferase, a 3-oxoadipyl-CoA
synthetase or a 3-oxoadipyl-CoA hydrolase; a 3-oxoadipate dehydrogenase; a 2-fumarylacetate
decarboxylase; a 3-oxopent-4-enoate reductase; a 3-hydroxypent-4-enoate dehydratase;
and a 2,4-pentadienoate decarboxylase;
(C) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA transferase, a 3-oxoadipyl-CoA
synthetase or a 3-oxoadipyl-CoA hydrolase; a 3-oxoadipate dehydrogenase; a 2-fumarylacetate
reductase; a 3-hydioxyhex-4-enedioate decarboxylase; and a 3-hydroxypent-4-enoate
decarboxylase;
(D) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA transferase, a 3-oxoadipyl-CoA
synthetase, or a 3-oxoadipyl-CoA hydrolase; a 3-oxoadipate dehydrogenase; a 2-fumarylacetate
reductase; a 3-hydroxyhex-4-enedioate decarboxylase; a 3-hydroxypent-4-enoate dehydratase;
and a 2,4-pentadienoate decarboxylase;
(E) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA transferase, a 3-oxoadipyl-CoA
synthetase, or a 3-oxoadipyl-CoA hydrolase; a 3-oxoadipate reductase; a 3-hydroxyadipate
dehydrogenase; a 3-hydroxyhex-4-enedioate decarboxylase; and a 3-hydroxypent-4-enoate
decarboxylase;
(F) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA transferase, a 3-oxoadipyl-CoA
synthetase or a 3-oxoadipyl-CoA hydrolase; a 3-oxoadipate reductase; a 3-hydroxyadipate
dehydrogenase; a 3-hydroxyhex-4-enedioate decarboxylase; a 3-hydroxypent-4-enoate
dehydratase; and a 2,4-pentadienoate decarboxylase;
(G) a succinyl-CoA:acetyl-CoA acyltransferase, a 3-oxoadipyl-CoA reductase; a 3-hydroxyadipyl-CoA
transferase, a 3-hydroxyadipyl-CoA synthetase, or a 3-hydroxyadipyl-CoA hydrolase;
a 3-hydroxyadipate dehydrogenase; a 3-hydroxyhex-4-enedioate decarboxylase; and a.
3-hydroxypent-4-enoate decarboxylase; and
(H) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA reductase; a 3-hydroxyadipyl-CoA
transferase, a 3-hydroxyadipyl-CoA synthetase, or a 3-hydroxyadipyl-CoA hydrolase;
a 3-hydroxyadipate dehydrogenase; a 3-hydroxyhex-4-enedioate decarboxylase; a 3-hydroxypent-4-enoate
dehydratase; and a 2,4-pentadienoate decarboxylase.
- 196. The non-naturally occurring microbial organism of item 193, wherein said microbial
organism further comprises:
- (i) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding
a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is
selected from an ATP-citrate lyase, a citrate lyase, a fumarate reductase, and an
alpha-ketoglutarate:ferredoxin oxidoreductase;
- (ii) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding
a reductive TCA pathway enzyme, wherein, said at least one exogenous nucleic acid
is selected from a pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase,
a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H2 hydrogenase; or
- (iii) at least one exogenous nucleic acid encodes an enzyme selected from a CO dehydrogenase,
an H2 hydrogenase, and combinations thereof.
- 197. The non-naturalty occurring microbial organism of item 196, wherein said microbial
organism comprising (i) further comprises an exogenous nucleic acid encoding an enzyme
selected from a pyruvate:ferredoxin oxidoreductase, an aconitase, an isocitrate dehydrogenase,
a succinyl-CoA synthetase, a succinyl-CoA transferase, a famarase, a malate dehydrogenase,
an acetate kinase, a phosphotransacetylase, an acetyl-CoA synthetase, an NAD(P)H:ferredoxin
oxidoreductase, ferredoxin, and combinations thereof.
- 198. The non-naturally occuring microbial organism of item 196, wherein said microbial
organism comprising (ii) further comprises an exogenous nucleic acid encoding an enzyme
selected from an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase,
a succinyl-CoA transferase, a. fumarase, a malate dehydrogenase, and combinations
thereof.
- 199. The non-naturally occurring microbial orgnaism of item 196, wherein said microbial
organism comprising (i) comprises four exogenous nucleic acids encoding an ATP-citrate
lyase, citrate lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin
oxidoreductase;
wherein said microbial organism, comprising (ii) comprises five exogenous nucleic
acids encoding a pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase,
a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H2 hydrogenase; or
wherein said microbial organism, comprising (iii) comprises two exogenous nucleic
acids encoding a CO dehydrogenase and an H2 hydrogenase.
- 200. The non-naturally occurring microbial organism of item 193, wherein, said at
least one exogenous nucleic acid is a heterologous nucleic acid,
- 201. The non-naturally occurring microbial organism of item 193, wherein said non-naturally
occurring microbial organism is in a substantially anaerobic culture medium.
- 202. A method for producing 1,3-butadiene, comprising culturing a non-naturally occurring
microbial organism of any one of items 193-201 under conditions and for a sufficient
period of time to produce 1,3-butadiene.
- 203. A non-naturally occurring microbial organism, comprising a microbial organism
having a 2,4-pentadienoate pathway comprising at least one exogenous nucleic acid
encoding a 2,4-pentadienoate pathway enzyme expressed in a sufficient amount to produce
2,4-pentadienoate, said 2,4-pentadienoate pathway selected from:
(A) a succinyt-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA transferase, a 3-oxoadipyl-CoA
synthetase ora 3-oxoadipyl-CoA hydrolase, a 3-oxoadipate dehydrogenase; a. 2-fumarylacetate
decarboxylase; a 3-oxopent-4-enoate reductase; and a 3-hydroxypent-4-enoate dehydratase;
(B) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA transferase, a 3-oxoadipyl-CoA
synthetase ora 3-oxoadipyl-CoA hydrolase; a 3-oxoadipate dehydrogenase; a 2-fumarylacetate
reductase; a 3-hydroxyhex-4-enedioate decarboxylase; and a 3-hydroxypent-4-enoate
dehydratase;
(C) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA transferase, a 3-oxoadipyl-CoA
synthetase ora 3-oxoadipyl-CoA hydrolase; a 3-oxoadipate reductase; a 3-hydroxyadipate
dehydrogenase; a 3-hydroxyhex-4-enedioate decarboxylase; and a 3-hydroxypent-4-enoate
dehydratase; and
(D) a succinyl-CoA:acetyl-CoA acyltransforase; a 3-oxoadipyl-CoA reductase; a 3-hydroxyadipyl-CoA
transferase, a 3-hydroxyadipyl-CoA synthetase or a 3-hydroxyadipyl-CoA hydrolase;
a 3-hydroxyadipate dehydrogenase; a 3-hydroxyhex-4-enedioate decarboxylase; and a
3-hydroxypent-4-enoate dehydratase.
- 204. The non-naturally occurring microbial organism of item 203, wherein said microbial
organism comprises two, three, four, five, or six exogenous nucleic acids each encoding
a 2,4-pentadienoate pathway enzyme.
- 205. The non-naturally occurring microbial organism of item 204, wherein said microbial
organism comprises exogenous nucleic acids encoding each of the enzymes selected from:
(A) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA transferase, a 3-oxoadipyl-CoA
synthetase, ora 3-oxoadipyl-CoA hydrolase; a 3-oxoadipate dehydrogenase; a 2-fumarylacetate
decarboxylase; a 3-oxopent-4-enoate reductase; and a 3-hydroxypent-4-enoate dehydratase;
(B) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA transferase, a 3-oxoadipyl-CoA
synthetase ora 3-oxoadipyl-CoA hydrolase; a 3-oxoadipate dehydrogenase; a 2-fumarylacetate
reductase; a 3-hydroxyhex-4-enedioate decarboxylase; and a 3-hydroxypent-4-enoate
dehydratase;
(C) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA transferase, a 3-oxoadipyl-CoA
synthetase ora 3-oxoadipyl-CoA hydrolase; a 3-oxoadipate reductase; a 3-hydroxyadipate
dehydrogenase; a 3-hydroxyhex-4-enedioate decarboxylase; and a 3-hydroxypent-4-enoate
dehydratase; and
(D) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA reductase; a 3-hydroxyadipyl-CoA
transferase, a 3-hydroxyadipyl-CoA synthetase or a 3-hydroxyadipyl-CoA hydrolase;
a 3-hydroxyadipate dehydrogenase; a 3-hydroxyhex-4-enedioate decarboxylase; and a
3-hydroxypent-4-enoate dehydratase.
- 206. The non-naturally occurring microbial organism of item 203, wherein said microbial
organism further comprises:
- (i) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding
a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is
selected from an ATP-citrate lyase, a citrate lyase, a fumarate reductase, and an
alpha-ketoglutarate:ferredoxin oxidoreductase;
- (ii) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding
a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is
selected from a pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase,
a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H2 hydrogenase; or
- (iii) at least, one exogenous nucleic acid encodes an enzyme selected from a CO dehydrogenase,
an H2 hydrogenase, and combinations thereof.
- 207. The non-naturally occurring microbial organism of item 206, wherein said microbial
organism comprising (i) further comprises an exogenous nucleic acid encoding an enzyme
selected from a pyruvate ferredoxin oxidoreductase, an aconitase, an isocitrate dehydrogenase,
a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase,
an acetate kinase, a phosphotransacetylase, an acetyl-CoA synthetase, an NAD(P)H:ferredoxin
oxidoreductase, ferredoxin, and combinations thereof.
- 208. The non-naturally occurring microbial organism of item 206, wherein said microbial
organism comprising (ii) further comprises at exogenous nucleic acid encoding an enzyme
selected from an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase,
a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, and combinations thereof.
- 209. The non-naturally occurring microbial orgnaism of item 206, wherein said microbial
organism comprising (i) comprises four exogenous nucleic acids encoding an ATP-citrate
lyase, citrate lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin
oxidoreductase;
wherein said microbial organism comprising (ii) comprises five exogenous nucleic acids
encoding a pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase,
a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H2 hydrogenase; or
wherein said microbial organism comprising (iii) comprises two exogenous nucleic acids
encoding a CO dehydrogenase and an H2 hydrogenase.
- 210. The non-naturally occurring microbial organism of item 203, wherein said at least
one exogenous nucleic acid is a heterologous nucleic acid.
- 211. The non-naturally occurring microbial organism of item 203, wherein said non-naturally
occurring microbial organism is in a substantially anaerobic culture medium.
- 212. A method for producing 2,4-pentadienoate, comprising culturing a non-naturally
occurring microbial organism, of any one of items 203-211 under conditions and for
a sufficient period of time to produce 2,4-pentadienoate.
- 213. A non-naturally occurring microbial organism, comprising a microbial organism
having a 1,3-butadiene pathway comprising at one exogenous nucleic acid encoding a
1,3-butadiene pathway enzyme expressed in a sufficient amount to produce 1,3-butadiene,
1,3-butadiene pathway selected from:
(A) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde
forming); a 3,5-dioxopentanoate reductase (aldehyde reducing); a 5-hydroxy-3-oxopentanoate
a 3,5-dihydroxypentanoate dehydratase; a 5-hydroxypent-2-enoate dehydratase; and a
2,4-pentadiene decarboxylase;
(B) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde
forming); a 3,5-dioxopentanoate reductase (aldehyde reducing); a 5-hydroxy-3-oxopentanoate
reductase; a 3,5-dihydroxypentanoate dehydratase; a 5-hydroxypent-2-enoate decarboxylase;
and a 3-butene-1-ol dehydratase;
(C) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde
forming); a 3,5-dioxopentanoate reductase (aldehyde reducing); a 5-hydroxy.3-oxopentanoate
reductase; a 3,5-dihydroxypentanoate decarboxylase; and a 3-butene-1-ol dehydratase;
(D) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (CoA reducing
and alcohol forming); a 5-hydroxy-3-oxopentanoatere reductase; a 3,5-dihydroxypentanoate
dehydratase; a 5-hydroxypent-2-enoate dehydratase and a 2,4-pentadiene decarboxylase;
(E) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (CoA reducing
and (alcohol forming); a 5-hydroxy-3-oxopentanoate reductase; a 3,5-dihydroxypentanoate
dehydratase; a 5-hydroxypent-2-enoate decarboxylase; and a 3-butene-1-ol dehydratase;
(F) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (CoA reducing
and alcohol forming); a 5-bydroxy-3-oxopentanoate reductase; a 3,5-dihydroxypentanoate
decarboxylase; and a 3-butene-1-ol dehydratase;
(G) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde
forming); a 3,5-dioxopentanoate reductase (ketone reducing); a 3-hydroxy-5-oxopentanoate
reductase; a 3,5-dihydroxypentanoate dehydratase; a 5-hydroxypent-2-enoate dehydratase;
and a 2,4-pentadiene decarboxylase;
(H) a malonyt-CoA.:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde
forming); a 3,5-dioxopentanoate reductase (ketone reducing); a 3-hydroxy-5-oxopentanoate
reductase; a 3,5-dihydroxypentanoate dehydratase; a 5-hydroxypent-2-enoate decarboxylase;
and a 3-butene-1-ol dehydratase;
(I) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde
forming); a 3,5-dioxopentanoate reductase (ketone reducing); a 3-hydroxy-5-oxopentanoate
reductase; a 3,5-dibydroxypentanoate decarboxylase; and a 3-butene-1-ol dehydratase;
(J) a malonyl-CoA:acetyl-CoA acyttransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing);
a 3-hydroxyglutaryt-CoA reductase (aldehyde forming); a 3-hydroxy-5-oxopentanoate
reductase; a 3,5-dihydroxypentanoate dehydratase; a 5-hydroxypent-2-enoate debydratase;
and a 2,4-pentadiene decarboxylase;
(K) a malonyl-CoAacetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing);
a 3-hydroxyglutaryl-CoA reductase (aldehyde forming); a 3-hydroxy-5-oxopentanoate,
reductase; a 3,5-dihydroxypentanoate dehydratase; a 5-hydroxypent-2-onoate decarboxylase;
and a 3-butene-1-ol dehydratase;
(L) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing);
a 3-hydroxyglutaryl-CoA reductase (aldehyde forming); a 3-hydroxy-5-oxopentanoate
reductase; a 3,5-dihydroxypentanoate decarboxylase; and a 3 -butene-1-ol dehydratase;
(M) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing);
a 3-hydroxyglutaryl-CoA reductase (alcohol forming); a 3,5-dihydroxypentanoate dehydratase;
a 5-hydroxypent-2-enoate dehydratase; and a 2,4-pentadiene decarboxylase;
(N) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-COA reductase (ketone-reducing);
a 3-hydroxyglutaryl-CoA reductase (alcohol forming); a 3,5-dihydroxypentanoate dehydratase;
a 5-hydroxypent-2-enoate decarboxylase; and a 3-butene-1-ol dehydratase; and
(O) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-feducing);
a 3-hydroxyglutaryl-CoA reductase (alcohol forming); a 3,5-dihydroxypentanoate decarboxylase;
and a 3-butene-1-ol dehydratase.
- 214. The non-naturally occurring microbial organism of item 213, wherein said microbial
organism comprises two, three, four, five, six or seven exogenous nucleic acids each
encoding a 1,3-butadiene pathway enzyme.
- 215. The non-naturally occurring microbial organism of item 214, wherein said microbial
organism comprises exogenous nucleic acids encoding each of the enzymes selected from:
(A) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-COA reductase (aldehyde
forming); a 3,5-dioxopentanoate reductase (aldehyde reducing); a 5-hydroxy-3-oxopentanoate
reductase; a 3,5-dihydroxypentanoate dehydratase; a 5-hydroxypent-2-enoate dehydratase;
and a 2,4-pentadiene decarboxylase;
(B) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde
forming); a 3,5-dioxopentanoate reductase (aldehyde reducing); a 5-hydroxy-3-oxopentanoate
reductase; a 3,5-dihydroxypentanoate dehydratase; a 5-hydroxypent-2-enoate decarboxylase;
and a 3-butene-1-ol dehydratase;
(C) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde
forming); a 3,5-dioxopentanoate reductase (aldehyde reducing); a 5-hydroxy-3-oxopentanoate
reductase; a 3,5-dihydroxypentanoate decarboxylase; and a 3-butene-1-ol dehydratase;
(D) a malonyl.COA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (CoA reducing
and alcohol forming); a 5-hydroxy-3-oxopentanoate reductase; a 3,5-dihydroxypentanoate
dehydratase; a 5-hydroxypent-2-enoate dehydratase; and a 2,4-pentadiene decarboxylase;
(E) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (CoA reducing
and alcohol, forming); a 5-hydroxy-3-oxopentanoate reductase; a 3,5-dihydroxypentanoate
dehydratase; a 5-hydroxypent-2-enoate decarboxylase; and a 3-butene-1-ol dehydratase;
(F) a malonyl-CoAacetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (CoA reducing
and alcohol forming); a 5-hydroxy-3-oxopentanoate reductase; a 3,5-dihydroxypentanoate
decarboxylase; and a 3-buteae-1-ol dehydratase;
(G) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde
forming); a 3,5-dioxopentanoate reductase (ketone reducing); a 3-hydroxy-5-oxopentanoate
reductase; a 3,5-dihydroxypentanoate dehydratase; a 5-hydroxypent-2-enoate dehydratase;
and a 2,4-pentadiene decarboxylase;
(H) a malonyl-CoA-acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde
forming); a 3,5-dioxopentanoate reductase (ketone reducing); a 3-hydroxy-5-oxopentanoate
reductase; a 3,5-dihydrroxypentanoate dehydratase; a 5-hydroxypent-2-enoate decarboxylase;
and a 3-butene-1-ol dehydratase;
(I) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde
forming); a 3,5-dioxopentanoate reductase (ketone reducing); a 3-hydroxy-5-oxopentanoate
reductase; a 3,5-dihydroxypentanoate decarboxylase; and a 3-butene-1-ol dehydratase;
(J) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing);
a 3-hydroxyglutaryl-CoA reductase (aldehyde forming); a 3-hydroxy-5-oxopentanoate
reductase; a 3,5-dihydroxypentanoate dehydratase; a 5-hydroxypent-2-enoate dehydratase;
and a 2,4-pentadiene decarboxylase;
(K) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing);
a 3-hydroxyglutaryl-CoA reductase (aldehyde forming); a 3-hydroxy-5-oxopentanoate
reductase; a 3,5-dihydroxypentanoate dehydratase; a 5-hydroxypent-2-enoate decarboxylase;
and a 3-butene-1-ol dehydratase;
(L) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing);
a 3-hydroxyglutaryl-CoA reductase (aldehyde forming); a 3-hydroxy-5-oxopentanoate
reductase; a 3,5-dihydroxypentanoate decarboxylase; and a 3-butene-1-ol dehydratase;
(M) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing);
a 3-hydroxyglutaryl-CoA. reductase (alcohol forming); a 3,5-dihydroxypentanoate dehydratase;
a 5-hydroxypent-2-enoate dehydratase; and a 2,4-pentadiene decarboxylase;
(N) a a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing);
a 3-hydroxyglutaryl-CoA reductase (alcohol forming); a 3,5-dihydroxypentanoate dehydratase;
a 5-hydroxypent-2-enoate decarboxylase; and a 3-butene-1-ol dehydratase; and
(O) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing);
a 3-hydroxyglutaryl-CoA reductase (alcohol forming); a 3,5-dihydroxypentanoate decarboxylase;
and a 3-butene-1-ol dehydratase.
- 216. The non-naturally occurring microbial organism of item 213, wherein said microbial
organism further comprises:
- (i) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding
a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is
selected from an ATP-citrate lyase, a citrate lyase, a fumarate reductase, and an
alpha-ketoglutarate:ferredoxin oxidoreductase;
- (ii) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding
a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is
selected from a pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase,
a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H2 hydrogenase; or
- (iii) at least one exogenous nucleic acid encodes an enzyme selected from a CO dehydrogenase,
an H2 hydrogenase, and combinations thereof.
- 217. The non-naturally occurring microbial organism of item 216, wherein said microbial
organism comprising (i) further comprises an exogenous nucleic acid encoding an enzyme
selected from a pyruvate:ferredoxin oxidoreductase, an aconitase, an isocitrate dehydrogenase,
a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a palate dehydrogenases,
an acetate kinase, a phosphotransacetylase, an acetyl-CoA synthetase, an NAD(P)H:ferredoxin
oxidoreductase, ferredoxin, and combinations thereof.
- 218. The non-naturally occurring microbial organism of item 216, wherein said microbial
organism comprising (ii) further comprises an exogenous nucleic acid encoding an enzyme
selected from an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase,
a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, and combinations thereof.
- 219. The non-naturally occurring microbial orgnaisin of item 216, wherein said microbial
organism comprising (i) comprises four exogenous nucleic acids encoding an ATP-citrate
lyase, citrate lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin
oxidoreductase;
wherein said microbial organism comprising (ii) comprises five exogenous nucleic acids
encoding a pyravate:ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase,
a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H2 hydrogenase; or
wherein said microbial organism comprising (iii) comprises two exogenous nucleic acids
encoding a CO dehydrogenase and an H2 hydrogenase.
- 220. The non-naturally occurring microbial organism of item 213, wherein said at least
one exogenous nucleic acid is a heterologous nucleic acid.
- 221. The non-naturally occurring microbial organism of item 213, wherein said non-naturally
occurring microbial organism is in a substantially anaerobic culture medium.
- 222. A method for producing 1,3-butadiene, comprising culturing a non-naturally occurring
microbial organism of any one of items 213-221 under conditions and for a sufficient
period, of time to produce 1,3-butadiene.
- 223. A non-naturally occurring microbial organism, comprising a microbial organism
having a 2,4-pentadienoate pathway comprising at least one exogenous nucleic acid
encoding a 2,4-pentadienoate pathway enzyme expressed in a sufficient amount to produce
2,4-pentadienoate, said 2,4-pentadienoate pathway selected from:
(A) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde
forming); a 3,5-dioxopentanoate reductase (aldehyde reducing); a 5-hydroxy-3-oxopentanoate
reductase; a 3,5-dihydroxypentanoate dehydratase; and a 5-hydroxypent-2-enoate dehydratase;
(B) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (CoA reducing
and alcohol forming); a 5-hydroxy-3-oxopeatanoate reductase; a 3,5-dihydroxypentanoate
dehydratase; and a 5-hydroxypent-2-enoate dehydratase;
(C) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde
forming); a 3,5-dioxopentanoate reductase (ketone reducing); a 3-hydroxy-5-oxopentanoate
reductase; a 3,5-dihydroxypentanoate dehydratase; and a 5-hydroxypent-2-enoate dehydratase;
(D) a matonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing);
a 3-hydroxyglutaryl-CoA reductase (aldehyde forming); a 3-hydroxy-5-oxopentanoate
reductase; a 3,5-dihydroxypentanoate dehydratase; and a 5-hydroxypent-2-enoate dehydratase;
and
(E) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing);
a 3-hydroxyglutaryl-CoA reductase (alcohol forming); a 3,5-dihydroxypentanoate dehydratase;
and a 5-hydroxypent-2-enoate dehydratase.
- 224. The non-naturally occurring microbial organism of item 223, wherein said microbial
organism comprises two, three, four, five or six exogenous nucleic acids each encoding
a 2,4-pentadienoate pathways enzyme.
- 225. The non-naturally occurring microbial organism of item 224, wherein said microbial
organism comprises exogenous nucleic acids encoding each of the enzymes selected from:
(A) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde
forming); a 3,5-dioxopentanoate reductase (aldehyde reducing); a 5-hydroxy-3-oxopentanoate
reductase; a 3,5-dihydroxypentanoate dehydratase; and a 5-hydroxypent-2-enoate dehydratase;
(B) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (CoA reducing
and alcohol forming); a 5-hydroxy-3-oxopentanoate reductase; a 3,5-dihydroxypentanoate
dehydratase; and a 5-hydroxypent-2-enoate dehydratase;
(C) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde
forming); a 3,5-dioxopentanoate reductase (ketone reducing); a 3-hydroxy-5-oxopentanoate
reductase; a 3,5-dihydroxypentanoate dehydratase; and a 5-hydroxypent-2-enoate dehydratase;
(D) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing);
a 3-hydroxyglutaryl-CoA reductase (aldehyde forming); a 3-hydroxy-5-oxopentanoate
reductase; a 3,5-dihydroxypentanoate dehydratase; and a 5-hydroxypent-2-enoate dehydratase;
and
(E) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reductag);
a 3-hydroxyglutaryl-CoA reductase (alcohol forming); a 3,5-dihydroxypentanoate dehydratase;
and a 5-hydroxypent-2-enoate dehydratase.
- 226. The non-naturally occurring microbial organism of item 223, wherein said microbial
organism further comprises:
- (i) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding
a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is
selected from an ATP-citrate lyase, a citrate lyase, a fumarate reductase, and an
alpha-ketoglutarate:ferredoxin oxidoreductase;
- (ii) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding
a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is
selected from a pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase,
a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H2 hydrogenase; or
- (iii) at least one exogenous nucleic acid encodes an enzyme selected from a CO dehydrogenase,
an H2 hydrogenase, and combinations thereof.
- 227. The non-naturally occurring microbial organism of item 226, wherein said microbial
organism comprising (i) further comprises an exogenous nucleic acid encoding an enzyme
selected from a pyruvate:terredoxin oxidoreductase, an aconitase, an isocitrate dehydrogenase,
a succinyl-CoA synthetase, a succinyl-CoA transferase, a furmase, a malate dehydrogenase,
an acetate kinase, a phosphotransacetylase, an acetyl-CoA synthetase, an NAD(P)H:ferredoxin
oxidoreductase, ferredoxin, and combinations thereof.
- 228. The non-naturally occurring microbial organism of item 226, wherein said microbial
organism comprising (ii) further comprises an exogenous nucleic acid encoding an enzyme
selected from an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase,
a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, and combinations thereof.
- 229. The non-naturally occurring, microbial orgnaism of item 226, wherein said microbial
organism comprising (i) comprises four exogenous nucleic acids encoding an ATP-citrate
lyase, citrate lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin
oxidoreductase;
wherein said microbial organism comprising (ii) comprises five exogenous nucleic acids
encoding a pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase,
a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H2 hydrogenase; or
wherein said microbial organism comprising (iii) comprises two erogenous nucleic acids
encoding a CO dehydrogenase and an H2 hydrogenase.
- 230. The non-naturally occurring microbial organism of item 223, wherein said at least
one exogenous nucleic acid is a heterologous nucleic acid.
- 231. The non-naturallly occurring microbial organism of item 223, wherein said non-naturally
occurring microbial organism is in a substantially anaerobic culture medium.
- 232. A method for producing 2,4-pentadienoate, comprising culturing a non-naturally
occurring microbial organism of any one of items 223-231 under conditions and for
a sufficient period of time to produce 2,4-pentadienoate.
- 233. A non-naturally occurring microbial organism, comprising a microbial organism
having a 3-butene-1-ol pathway comprising at least one exogenous nucleic acid encoding
a 3-butene-1-ol pathway enzyme expressed in a sufficient amount to produce 3-butene-1-ol,
said 3-butene-1-ol pathway selected from:
(A) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-COA reductase (aldehyde
forming), a 3,5-dioxopentanoate reductase (aldehyde reducing); a 5-hydroxy-3-oxopentanoate
reductase; a 3,5-dihydroxypentanoate dehydratase; and a 5-hydroxypent-2-enoate decarboxylase;
(B) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde
forming), a 3,5-dioxopentanoate reductase (aldehyde reducing); a 5-hydroxy-3-oxopentanoate
reductase; and a 3,5-dihydroxypentanoate docarboxylase;
(C) a matonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (CoA reducing
and alcohol forming); a 5-hydroxy-3-oxopentanoate reductase; a 3,5-dihydroxypentanoate
dehydratase; and a 5-hydroxypent-2-enoate decarboxylase;
(D) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (CoA reducing
and alcohol forming); a 5-hydroxy-3-oxopentanoate reductase; and a 3,5-dihydroxypentanoate
decarboxylase;
(E) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde
forming); a 3,5-dioxopentanoate reductase (ketone reducing); a. 3-hydroxy-5-oxopentanoate
reductase; a 3,5-dihydroxypentanoate dehydratase; and a 5-hydroxypent-2-enoate decarboxylase;
(F) a malonyl-COA:acetyl-COA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde
forming); a 3,5-dioxopentanoate reductase (ketone reducing); a 3-hydroxy-5-oxopentanoate
reductase; and a 3,5-dihydroxypentanoate decarboxylase;
(G) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing);
a 3-hydroxyglutaryl-CoA reductase (aldehyde forming); a 3-hydroxy-5-oxopentanoate
reductase; a 3,5-dihydroxypentanoate dehydratase; and a 5-hydroxypent-2-enoate decarboxylase;
(H) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing);
a 3-hydroxyglutaryl-CoA reductase (aldehyde forming); a 3-hydroxy-5-oxopentanoate
reductase; and a 3,5-dihydroxypentanoate decarboxylase;
(I) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxogluraryl-CoA reductase (ketone-reducing);
a 3-hydroxyglutaryl-CoA reductase (alcohol forming); a 3,5-dihydroxypentanoate dehydratase;
and a 5-hydroxypent-2-enoate decarboxylase; and
(J) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing);
a 3-hydroxyglutaryl-CoA reductase (alcohol forming); and a 3,5-dihydroxypentanoate
docarboxylase.
- 234. The non-naturally occurring microbial organism of item 233, wherein said microbial
organism comprises two, three, four or five exogenous nucleic acids each encoding
a 3-butene-1-ol pathway enzyme.
- 235. The non-naturally occurring microbial organism of item 234, wherein said microbial
organism comprises exogenous nucleic acids encoding each of the enzymes selected from:
(A) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde
forming); a 3,5-dioxopentanoate reductase, (aldehyde reducing); a 5-hydroxy-3-oxopentanoate
reductase; a 3,5-dihydroxypentanoate dehydratase; and a 5-hydroxypent-2-enoate decarboxylase;
(B) a matonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde
forming); a 3,5-dioxopentanoate reductase (aldehyde reducing); a 5-hydroxy-3-oxopentanoate
reductase; and a 3,5-dihydroxypentanoate decarboxytase;
(C) a malonyl-Co.A:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (CoA
reducing and alcohol forming); a 5-hydroxy-3-oxopentanoate reductase; a 3,5-dihydroxypentanoate
dehydratase; and a 5-hydroxypent-2-enoate decarboxylase;
(D) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglatatyl-CoA reductase (CoA reducing
and alcohol forming); as 5-hydroxy-3-oxopentanoate reductase; and a 3,5-dihydroxypentanoate
decarboxylase;
(E) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde
forming); a 3,5-dioxopentanoate reductase (ketone reducing); a 3-hydroxy-5-oxopentanoate
reductase; a 3,5-dihydroxypentanoate dehydratase; and a 5-hydroxypent-2-enoate decarboxylase;
(F) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde
forming); a 3,5-dioxopentanoate reductase (ketone reducing); a 3-hydroxy-5-oxopentanoate
reductase; and a 3,5-dihydroxypentanoate decarboxylase;
(G) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing);
a 3-hydroxyglutaryl-CoA reductase (aldehyde forming); a 3-hydroxy-5-oxopentanoate
reductase; a 3,5-dihydroxypentanoate dehydratase; and a 5-hydroxypent-2-enoate decarboxylase;
(H) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing);
a 3-hydroxyglutaryl-CoA (aldehyde forming); a 3-hydroxy-5-oxopentanoate reductase;
and a 3,5-dihydroxypentanoate decarboxylase;
(I) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxogtutaryl-CoA reductase (ketone-reducing);
a 3-hydroxyglutaryl-CoA reductase (alcohol forming); a 3,5-dihydroxypentanoate dehydratase;
and a 5-hydroxypent-2-enoate decarboxylase; and
(J) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing);
a 3-hydroxyglutaryl-CoA reductase (alcohol forming); and a 3,5-dihydroxypentanoate
decarboxylase.
- 236. The non-naturally occurring microbial organism of item 233, wherein said microbial
organism further comprises:
(i) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding
a reductive TCA pathway enzyme, wherein, said at least one exogenous nucleic acid
is selected from an ATP-citrate lyase, a citrate lyase, a fumarate reductase, and
an alpha-ketoglutarate:ferredoxin oxidoreductase;
(ii) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding
a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is
selected from a pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase,
a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H2 hydrogenase; or
(iii) at least one exogenous nucleic acid encodes an enzyme selected from a CO dehydrogenase,
in H2 hydrogenase, and combinations thereof.
- 237. The non-naturally occurring microbial organism of item 236, wherein said microbial
organism comprising (i) further comprises an exogenous nucleic acid encoding an enzyme
selected from a pyruvate:ferredoxin oxidoreductase, an aconitase, an isocitrate dehydrogenase,
a succinyl-CoA synthetase, a succinyl-CoA a fumarase, a malate dehydrogenase, an kinase,
a phosphotransacetylase, an acetyl-CoA synthetase, an NAD(P)H:ferredoxin oxidoreductase,
ferredoxin, and combinations thereof.
- 238. The non-naturally occurring microbial organism of item 236, wherein said microbial
organism comprising (ii) further comprises an exogenous nucleic acid encoding an enzyme
selected from an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase,
a succinyl-COA transferase, a fumarase, a malate dehydrogenase, and combinations thereof.
- 239. The non-naturally occurring microbial orgnaism of item 236, wherein, said microbial
organism comprising (i) comprises four exogenous nucleic acids encoding an ATP-citrate
lyase, citrate lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin
oxidoreductase;
wherein said microbial organism comprising (ii) comprises five exogenous nucleic acids
encoding a pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase,
a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H2 hydrogenase; or
wherein said microbial organism comprising (iii) comprises two exogenous nucleic encoding
a CO dehydrogenase and an H2 hydrogenase.
- 240. The non-naturally occurring microbial organism of item 233, wherein said at least
one exogenous nucleic acid is a heterologous nucleic acid.
- 241. The non-naturally occurring microbial organism of item 233, wherein said non-naturally
occurring microbial organism, is in a substantially anaerobic culture medium.
- 242. A method for producing 3-butene-1-ol, comprising culturing a non-naturally occurring
microbial organism of any one of items 233-241. under conditions and for a sufficient
period of time to produce 3-butene-1-ol.
- 243. A method for producing 1,3-butadiene comprising, culturing a non-naturally occurring
microbial organism of any one of items 233-241 under conditions and for a sufficient
period of time to produce 3-butene-1-ol, and chemically converting said 3-butene-1-ol
to 1,3-butadiene.